Site-specific glycoengineering of targeting moieties

ABSTRACT

The current disclosure provides binding polypeptides (e.g., antibodies), and targeting moiety conjugates thereof, comprising a site-specifically engineered glycan linkage within native or engineered glycans of the binding polypeptide. The current disclosure also provides nucleic acids encoding the antigen-binding polypeptides, recombinant expression vectors and host cells for making such antigen-binding polypeptides. Methods of using the antigen-binding polypeptides disclosed herein to treat disease are also provided.

RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.14/662,187, filed Mar. 18, 2015, which claims the benefit of priority ofU.S. Provisional Patent Application Nos. 61/955,682, filed Mar. 19,2014, and 62/061,556, filed Oct. 8, 2014. The contents of theaforementioned applications are hereby incorporated by reference intheir entirety for all purposes.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Mar. 29, 2021, isnamed 715712_SA9-148DIV_ST25.txt and is 115,004 bytes in size.

BACKGROUND

Use of specific antibodies to treat people and other animals is apowerful tool that has been very effective in treating many conditionsand disorders. However, there is great demand for more effectivetargeted therapeutics, especially target specific therapies with higherefficacy and greater therapeutic windows. One of these target specifictreatments employs antibody-effector moiety conjugates in which atargeting moiety directs a specific antibody to a desired treatmentsite. These molecules have shown improved therapeutic index—higherefficacy and/or lower toxicity profiles than the un-targeted antibody ina clinical setting. However, development of such therapeutics can bechallenging as many factors, including physical and/or structuralproperties of the antibody itself as well as linkage stability, can havesignificant impact on the disease target (e.g. tumor) specificity,thereby reducing efficacy. With high non-specific binding and lowstability in circulation, the antibody-effector moiety conjugate istypically cleared through normal tissues before reaching the targetsite. Moreover, antibody-effector moiety conjugates with significantsubpopulations of high drug loading could generate aggregates whichwould be eliminated by macrophages, leading to shorter half-life. Thus,there are increasing needs for critical process control and improvementas well as preventing complications, such as antibody aggregation andnonspecific antibody-mediated toxicity.

Although antibody-effector moiety conjugates generated according tocurrent methods can be effective, development of such therapeutics arechallenging, as heterogeneous mixtures are often a consequence of theconjugation chemistries used. For example, effector moiety conjugationto antibody lysine residues is complicated by the fact that there aremany lysine residues (˜30) in an antibody available for conjugation.Since the optimal number of conjugated effector moiety to antibody ratio(DAR) is much lower to minimize loss of function of the antibody (e.g.,around 4:1), lysine conjugation often generates a very heterogeneousprofile. Furthermore, many lysines are located in critical antigenbinding sites of the CDR region, and drug conjugation may lead to areduction in antibody affinity. Thiol mediated conjugation mainlytargets the eight cysteines involved in hinge disulfide bonds. However,it is still difficult to predict and identify which four of eightcysteines are consistently conjugated among the different preparations.More recently, genetic engineering of free cysteine residues has enabledsite-specific conjugation with thiol-based chemistries, but suchlinkages often exhibit highly variable stability, with the linkerundergoing exchange reactions with albumin and other thiol-containingserum molecules. Therefore, a site-specific conjugation strategy whichgenerates an antibody conjugate with a defined conjugation site andstable linkage would be useful to enable effector moiety conjugationwhile minimizing adverse effects on antibody structure or function.

SUMMARY

The current disclosure provides binding polypeptides (e.g., antibodies),and targeting moiety conjugates thereof. In certain embodiments, theconjugates comprise a site-specifically engineered targeting-moietyglycan linkage within native or modified glycans of the bindingpolypeptide. The current disclosure also provides nucleic acid sequencesencoding the antigen-binding polypeptides, recombinant expressionvectors, and host cells for making such antigen-binding polypeptides.Methods of using the antigen-binding polypeptides disclosed herein totreat disease are also provided.

Accordingly, in one aspect, the invention provides a binding polypeptidecomprising at least one modified glycan comprising at least one moietyof Formula (IV):

wherein:

A) Q is NH or O;

B) CON is a connector moiety;C) X is a targeting moiety;D) Gal is a component derived from galactose; andE) Sia is a component derived from sialic acid;wherein Sia is present or absent, and wherein the targeting moiety bindsto a cell.

In one embodiment, the cell is a mammalian cell. In a furtherembodiment, the cell is selected from an immune cell, a liver cell, atumor cell, a vascular cell, an epithelial cell, or a mesenchymal cell.In yet another embodiment, the cell is selected from a B cell, a T cell,a dendritic cell, a natural killer (NK) cell, a macrophage, aneutrophil, a hepatocyte, a liver sinusoidal endothelial cell, or ahepatoma cell.

In one embodiment, the binding polypeptide is internalized by the cell.In another embodiment, the amount of the binding polypeptideinternalized by the cell is greater than the amount of a referencebinding polypeptide lacking a targeting moiety internalized by the cell.

In one embodiment, the targeting moiety binds to a mannose 6 phosphatereceptor on the cell. In another embodiment, the targeting moietycomprises a mannose 6 phosphate (Man 6-P) moiety.

In one embodiment, the targeting moiety binds to a Siglec on the cell.In a further embodiment, the Siglec is sialoadhesin (Siglec-1), CD22(Siglec-2), CD33 (Siglec-3), MAG (Siglec-4), Siglec-5, Siglec-6,Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12,Siglec-14, or Siglec-15. In another embodiment, the targeting moietycomprises an α2,3-, α2,6-, or α2,8-linked sialic acid residue. In afurther embodiment, the targeting moiety comprises an α2,3-siallylactosemoiety or an α2,6-siallylactose moiety.

In one embodiment, the targeting moiety binds to a C-type lectinreceptor, a galectin, a L-type lectin receptor or other carbohydratereceptors. In a further embodiment, the targeting moiety binds toDEC-205 (CD205; lymphocyte antigen 75), macrophage mannose receptor(MMR; CD206), Dectin-1, Dectin-2, macrophage-inducible C-type lectin(Mincle), dendritic cell-specific ICAM3-grabbing nonintegrin (DC-SIGN;CD209), DC NK lectin group receptor-1 (DNGR-1), Langerin (CD207), alectican, an asialoglycoprotein receptor (ASGPR), C-lectin receptordendritic cell immunoreceptor (CLEC4A; CLECSF6; DCIR), macrophagegalactose-type lectin (MGL), a DC receptor, a collectin, a selectin, anNK-cell receptor, a multi-C-type lectin domain (CTLD) endocyticreceptor, a Reg group (type VII) lectin, chondrolectin, tetranectin,polycystin, attractin (ATRN), eosinophil major basic protein (EMBP),DiGeorge Syndrome Critical Region Gene 2 (DGCR2), Thrombomodulin,Bimlec, a group XVI lectin (SEEC), or a group XVII lectin(CBCP/Frem1/QBRICK).

In one embodiment, Q is O.

In one embodiment, the glycan comprises at least one moiety of thefollowing structural formula:

In an embodiment, the targeting moiety is a trivalent GalNAc glycanmoiety.

In one embodiment, the targeting moiety is a glycopeptide. In a furtherembodiment, the targeting moiety is a tri-galactosylated glycopeptide,e.g., lactose₃-Cys₃Gly₄.

In an embodiment, Sia is present and the lactose₃-Cys₃Gly₄moiety isrepresented by Formula V:

In one embodiment, the glycan comprises at least one moiety of thefollowing structural formula:

In one embodiment, the glycan comprises at least one moiety of thefollowing structural formula:

In one embodiment, the targeting moiety is a trivalent GalNAc glycanmoiety.

In one embodiment, Sia is present and the trivalent GalNAc glycan moietyis represented by Formula VIII:

[Formula VIII], wherein q is an integer between 1 and 29 inclusive.

In another embodiment, the glycan comprises at least one moiety havingthe following structural formula:

wherein q is an integer between 1 and 29 inclusive.

In another embodiment, the glycan comprises at least one moiety havingthe following structural formula:

wherein q is an integer between 1 and 29 inclusive.

In an embodiment, the trivalent GalNAc glycan moiety is represented byFormula VII, Formula XIII, or Formula XIV:

In one embodiment, the glycan comprises at least one moiety selectedfrom the following structural formulae:

In one embodiment, the glycan comprises at least one moiety selectedfrom the following structural formulae:

In an embodiment, the binding polypeptide comprises an Fc domain. In afurther embodiment, the modified glycan is N-linked to the bindingpolypeptide via an asparagine residue at amino acid position 297 of theFc domain, according to EU numbering. In another embodiment, themodified glycan is N-linked to the binding polypeptide via an asparagineresidue at amino acid position 298 of the Fc domain, according to EUnumbering.

In one embodiment, the Fc domain is human. In another embodiment, thebinding polypeptide comprises a CH1 domain. In a further embodiment, themodified glycan is N-linked to the binding polypeptide via an asparagineresidue at amino acid position 114 of the CH1domain, according to Kabatnumbering.

In one embodiment, the connector moiety comprises a pH-sensitive linker,disulfide linker, enzyme-sensitive linker or other cleavable linkermoiety. In an embodiment, the connector moiety comprising a linkermoiety selected from the group of linker moieties depicted in Table 2 or14.

In one embodiment, the binding polypeptide is an antibody orimmunoadhesin.

In one aspect, the present invention provides a method of making thebinding polypeptide of any one of the preceding claims, the methodcomprising reacting an effector moiety of Formula (I):

NH₂-Q-CON—X  Formula (I),

wherein:

A) Q is NH or O;

B) CON is a connector moiety; and

C) X is a targeting moiety,

with an precursor binding polypeptide comprising an oxidized glycan.

In one embodiment, the initial binding polypeptide comprises at leastone moiety of the following structural formula:

In one embodiment, the initial binding polypeptide comprises at leastone moiety of the following structural formula:

In one embodiment, the precursor binding polypeptide comprises anoxidized glycan comprises at least one moiety of the followingstructural formula:

In one embodiment, the precursor binding polypeptide comprises anoxidized glycan comprises at least one moiety of the followingstructural formula:

In one embodiment, the precursor binding polypeptide comprising anoxidized glycan is generated by reacting an initial binding polypeptidecomprising a glycan with a mildly oxidizing agent. In a furtherembodiment, the mildly oxidizing agent is sodium periodate. In anotherembodiment, no more than 1 mM sodium periodate is employed. In anotherembodiment, the mildly oxidizing agent is galactose oxidase.

In one embodiment, the method of marking a binding protein comprisesreacting an effector moiety of the following structural formula:

NH₂—O—CON—X

with the precursor binding polypeptide comprising an oxidized glycancomprising at least one moiety of the following structural formula:

to form a binding polypeptide of the following structural formula:

In an embodiment, the targeting moiety is a trivalent GalNAc glycanmoiety. In an embodiment, the reacting step is conducted in the presenceof a salt comprising a metal ion. In a further embodiment, wherein themetal ion is a copper ion. In one embodiment, the salt is copperacetate. In another embodiment, the salt comprising a metal ion ispresent at a concentration of at least 0.1 mM.

In another embodiment, the binding polypeptide comprising the glycancomprises one or two terminal sialic acid residues. In a furtherembodiment, the terminal sialic acid residues are introduced bytreatment of the binding polypeptide with a sialyltransferase orcombination of sialyltransferase and galactosyltransferase. In oneembodiment, an initial binding polypeptide is contacted with a mildlyoxidizing agent in order to produce a precursor binding polypeptide orprecursor binding protein. In one embodiment, the precursor bindingpolypeptide or precursor binding protein comprises an oxidized sialicacid moiety comprising a terminal aldehyde.

In one aspect, the present invention provides a binding polypeptidecomprising at least one modified glycan comprising at least one moietyof Formula (IV):

wherein:

A) Q is NH or O;

B) CON is a connector moiety; andC) X is a lactose₃-Cys₃Gly₄ moiety;D) Gal is a component derived from galactose;E) Sia is a component derived from sialic acid; andwherein Sia is present and the lactose₃-Cys₃Gly₄moiety is represented byFormula V:

In another embodiment, the present invention provides a compositioncomprising a binding polypeptide described supra and a pharmaceuticallyacceptable carrier or excipient. In one embodiment, the ratio oftargeting moiety to binding polypeptide is equal to or more than about4. In another embodiment, the ratio of targeting moiety to bindingpolypeptide is at least about 2. In a further embodiment, the presentinvention provides a method for treating a patient in need thereofcomprising administering an effective amount of the composition.

In one aspect, the present invention provides a binding polypeptidecomprising at least one modified glycan comprising at least one moietyof Formula (IV):

wherein:

A) Q is NH or O;

B) CON is a connector moiety; andC) X is a moiety comprising PEG;D) Gal is a component derived from galactose; andE) Sia is a component derived from sialic acid;wherein Sia is present or absent.

In one embodiment, the glycan comprises at least one moiety representedby Formula IX or Formula XI:

wherein p has a value of 1 to 32; or

wherein p has a value of 1 to 32.

In an embodiment, the glycan comprises at least one moiety selected fromthe following structural formulae:

wherein p has a value of 1 to 32; or

wherein p has a value of 1 to 32.In one embodiment, the glycan comprises at least one moiety selectedfrom the following structural formulae:

wherein p has a value of 1 to 32; or

wherein p has a value of 1 to 32.

In an embodiment, the targeting moiety comprises PEG and is representedby Formula X or Formula XII:

In one embodiment, the glycan comprises at least one moiety selectedfrom the following structural formulae:

In an embodiment, the glycan comprises at least one moiety selected fromthe following structural formulae:

In one embodiment, the PEG moiety comprises mono-PEG, bi-PEG, ortri-PEG. In another embodiment, the PEG moiety comprises 3 to 3.5 PEG.In another embodiment, the binding polypeptide is an antibody orimmunoadhesin.

In another aspect, the present invention provides a method of making aPEGylated binding polypeptide comprising at least one oxidized glycan,wherein the method comprises: (a) reacting a binding polypeptidecomprising at least one glycan with a mildly oxidizing agent in thepresence of a salt comprising a metal ion, and (b) conjugating theoxidized binding polypeptide with at least one moiety comprising PEG.

In one embodiment, the metal ion is a copper ion. In another embodiment,the salt is copper acetate. In another embodiment, the salt comprising ametal ion is present at a concentration of at least 0.1 mM. In anotherembodiment, the mildly oxidizing agent is periodate or galactoseoxidase. In another embodiment, the at least one glycan is a modifiedglycan.

In one embodiment, the method of making a binding polypeptide comprises:a) reacting a binding polypeptide comprising at least one modifiedglycan with a mildly oxidizing agent in the presence of a saltcomprising a metal ion; and b) conjugating the oxidized bindingpolypeptide with the moiety comprising PEG. In a further embodiment, thePEG moiety comprises mono-PEG, bi-PEG, or tri-PEG.

In another aspect, the present invention provides a binding polypeptidecomprising at least one modified glycan comprising at least one moietyof Formula (IV):

wherein:

A) Q is NH or O;

B) CON is a connector moiety; andC) X is a trivalent GalNAc glycan;D) Gal is a component derived from galactose; andE) Sia is a component derived from sialic acid;wherein Sia is present and the trivalent GalNAc glycan moiety isrepresented by Formula VI.

In one embodiment, the binding protein comprises an N-glycan glycoformselected from the group consisting of: a G0 glycoform, a G1 glycoform,and a G2 glycoform. In a further embodiment, the N-glycan glycoform isselected from the group consisting of: a G1S1 glycoform, a G2S1glycoform, a G2S2 glycoform, a G1F glycoform, a G2F glycoform, a G1S1Fglycoform, a G2S1F glycoform, and a G2S2F glycoform.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings. The patent or application file contains at least one drawingexecuted in color. Copies of this patent or patent applicationpublication with color drawing(s) will be provided by the Office uponrequest and payment of the necessary fee.

FIGS. 1A-1C are a schematic illustration of the synthesis of an antibodydrug conjugate where a toxin moiety is linked to an oxidized sialic acidresidue of the antibody glycan using an oxime linkage. FIG. 1A depictsantibodies with wild type (existing) carbohydrates at position Asn297and with engineered glycosylation sites at positions 114 (according tothe Kabat numbering) and 298 (according to the EU numbering system).FIG. 1B depicts both the canonical glycan structure G1F (left, seen atposition Asn297 on the antibodies shown in FIG. 1A) and the G2S1Fstructure (right, seen at positions Ala114 and Asn298 on the antibodiesshown in FIG. 1A). The glycan structures shown in FIG. 1B have severaldifferent subunits: the white square boxes represent GlcNAc, theupside-down white triangle represents fucose, the white circlesrepresent mannose, the shaded circles represent galactose, and theshaded upright triangle represents sialic acid. FIG. 1C depicts thepreparation of a conjugate of the instant invention. On the left is aninitial binding protein with a Gal and Sia moiety. Following oxidationwith NaIO4, the sialic moiety is oxidized to form a precursor bindingprotein and then reacted with a toxin-containing moiety to form abinding peptide comprising at least one moiety of Formula IV.

FIG. 2 is a Coomassie-blue stained gel showing the expression andpurification of glycosylation mutants.

FIG. 3 depicts the results of surface plasmon resonance experiments usedto assess the binding of α|3TCR HEBE1 IgG antibody mutants torecombinant human FcγRIIIa (V158 & F158).

FIG. 4 depicts the results of surface plasmon resonance experiments usedto assess the binding of αβTCR HEBE1 IgG antibody mutants to recombinanthuman FcγRI.

FIG. 5 depicts the cytokine release profile from PBMCs for TNFa, GM-CSF,IFNy and IL10 in the presence of mutant anti-αβTCR antibodies (day 2).

FIG. 6 depicts the cytokine release profile from PBMCs for IL6, IL4 andIL2 in the presence of mutant anti-αβTCR antibodies (day 2).

FIG. 7 depicts the cytokine release profile from PBMCs for TNFa, GM-CSF,IFNy and IL10 in the presence of mutant anti-αβTCR antibodies (day 4).

FIG. 8 depicts the cytokine release profile from PBMCs for IL6, IL4 andIL2 in the presence of mutant anti-αβTCR antibodies (day 4).

FIGS. 9A-9B depict the results of experiments investigating theexpression level of 2C3 mutants by Western blotting (FIG. 9A) andsurface plasmon resonance (FIG. 9B).

FIG. 10 depicts the results of experiments investigating glycosylationof 2C3 mutants pre- and post-PNGase F treatment.

FIG. 11 depicts the results of SDS-PAGE experiments investigatingglycosylation sites on 2C3 mutants isolated from cell culture.

FIGS. 12A-12C depict the results of surface plasmon resonanceexperiments used to assess the binding of modified anti-CD52 torecombinant human FcγRIIIa (V158). Anti-CD52 comprising S298N/Y300Smutations in the Fc domain were used to assess the effector function ofthe modified molecule. binding to CD52 peptide (FIG. 12A), binding toFcγRIIIa (V158, FIG. 12B), and control binding to mouse FcRn (FIG. 12C).

FIG. 13 depicts the results of surface plasmon resonance experimentsinvestigating the Fc binding properties of 2C3 mutants.

FIGS. 14A-14B depict the results of surface plasmon resonanceexperiments investigating the binding of modified anti-CD52 to bothFcγRIIIa (Val158) (as above) and FcγRIIIa (Phe158). Anti-CD52 antibodiescomprising S298N/Y300S mutations in the Fc domain were used to assessthe effector function of the modified molecule binding to FcγRIIIa(Val158, FIG. 14A) and FcγRIIIa (Phe58, FIG. 14B).

FIGS. 15A-15B depict the analysis of C1q binding in the S298N/Y300Smutant and the WT 2C3 control (FIG. 15A) and the results of an Elizaanalysis confirming equivalent coating of the wells (FIG. 15B).

FIG. 16 depicts the results of plasmon resonance experiments experimentsmeasuring the binding kinetics of 2C3 mutants to CD-52 peptide 741.

FIG. 17 depicts the results of plasmon resonance experiments experimentscomparing the antigen binding affinity of WT anti-CD-52 2C3 and theA114N hyperglycosylation mutant.

FIGS. 18A-18D depict the results of isoelectric focusing and massspectrometry charge characterization experiments to determine the glycancontent of 2C3 mutants.

FIGS. 19A-19B depict the results of concentration (Octet) and plamonresonance experiments comparing the antigen binding affinity of WTanti-CD52 2C3 and mutants.

FIG. 20 depicts the results of SDS-PAGE experiments to demonstrate theadditional glycosylation of the anti-TEM1 A114N mutant.

FIG. 21 depicts the results of SDS-PAGE and hydrophobic interactionchromatography analysis of the A114N anti-Her2 mutant.

FIG. 22 depicts the results of SDS-PAGE experiments to demonstrate theconjugation of PEG to the 2C3 A114N mutant through an aminooxy linkage.

FIG. 23 depicts the results of LC-MS experiments to determine the glycancontents of anti-TEM1 A114N hyperglycosylation mutant.

FIG. 24 depicts the results of LC-MS experiments to determine the glycancontents of a wild-type HER2 antibody and an A114N anti-Her2hyperglycosylation mutant.

FIGS. 25A-25C depict an exemplary method for performing site-specificconjugation of an antibody.

FIG. 26 depicts a synthesis of exemplary effector moieties:aminooxy-Cys-MC-VC-PABC-MMAE and aminooxy-Cys-MC-VC-PABC-PEG8-Dol10.

FIGS. 27A-27C depict characterization information for a sialylated HER2antibody.

FIGS. 28A-28D depict characterization information for oxidizedsialylated anti-HER 2 antibody.

FIG. 29 depicts hydrophobic interaction chromatographs ofglycoconjugates prepared with three different sialylated antibodies withtwo different aminooxy groups.

FIG. 30 shows a HIC chromatograph of antiHer2 A114 glycosylation mutantconjugate with AO-MMAE prepared using GAM(+) chemistry.

FIGS. 31A-31D depict a comparison of the in vitro potency of ananti-HER2 glycoconjugate and thiol conjugate.

FIG. 32 depicts a comparison of the in vitro potency of an anti FAP B11glycoconjugate and thiol conjugate.

FIGS. 33A-33D depict a comparison of in vivo efficacy of anti-HER2glycoconjugates and thiol conjugates in a Her2+ tumor cell xenograftmodel.

FIG. 34 depicts the results of LC-MS experiments to determine the glycancontent of a mutant anti-αβTCR antibody containing the S298N/Y300Smutation.

FIG. 35 depicts the results of circular dichroism experiments todetermine the relative thermal stability of a wild-type anti-αβTCRantibody and mutant anti-αβTCR antibody containing the S298N/Y300Smutation.

FIG. 36 depicts the results of a cell proliferation assay for ADCprepared with the anti-HER antibody bearing the A114N hyperglycosylationmutation and AO-MMAE.

FIG. 37 is a schematic illustration of the synthesis of an antibody drugconjugate where a targeting moiety is linked to an oxidized sialic acidresidue of the antibody glycan using an oxime linkage.

FIG. 38 is a schematic illustration depicting an exemplary method forperforming site-specific conjugation of an antibody to a glycopeptidethrough an oxime linkage according to the described methods.

FIG. 39 is a schematic illustration depicting site-specific conjugationof neoglycans to antibody through sialic acid in native Fc glycans.

FIG. 40 is a series of exemplary glycans that may be used forconjugation including lactose aminooxy and bis Man-6-P (or bisM6P)hexamannose aminooxy (for aminooxy conjugation).

FIG. 41 is a schematic depiction of the preparation of Man-6-Phexamannose maleimide.

FIG. 42 depicts SDS-PAGE and MALDI-TOF characterization of Man-6-Phexamannose aminooxy conjugates made with rabbit polyclonal antibody.

FIG. 43 depicts the results of surface plasmon resonance experimentsused to assess the binding of control and Man-6-P hexamannose conjugatedrabbit IgG antibodies to Man-6-P receptor.

FIG. 44 depicts the uptake of Man-6-P conjugated rabbit IgG antibody inHepG2 and RAW cells.

FIG. 45 depicts the characterization of control, Man-6-P conjugated, andlactose conjugated antibodies through SDS-PAGE and lectin blotting.

FIG. 46 depicts the results of MALDI-TOF intact protein analyses forcontrol, Man-6-P conjugated, and lactose conjugated antibodies.

FIG. 47 depicts the characterization of polyclonal antibody conjugatedto Man-6-P hexamannose maleimide (thiol conjugation at hinge cysteines)through SDS-PAGE (non-reducing and reducing), lectin blot (reducing),and Man-6-P quantitation.

FIG. 48 depicts the characterization of polyclonal antibody conjugatedto lactose maleimide (thio conjugation at hinge cysteines) throughSDS-PAGE and galactose quantitation.

FIG. 49 depicts the characterization of monoclonal antibody conjugatedto Man-6-P hexamannose maleimide (thiol conjugation at hinge cysteines)through SDS-PAGE (non-reducing and reducing), and glycan (bis Man-6-P)quantitation.

FIG. 50 depicts the results of size exclusion chromatography (SEC)analysis of a hinge cysteine polyclonal antibody conjugate.

FIG. 51 depicts the results of size exclusion chromatography (SEC)analysis of a hinge cysteine monoclonal antibody conjugate.

FIG. 52 depicts the results of sialidase titration and sialic acidquantitation to determine the amount of sialic acid release from NNAS,sialylated NNAS, and desialylated and galatosylated NNAS antibodies.

FIG. 53 depicts the results of MALDI-TOF MS analysis to determine theglycan structures of a mouse NNAS antibody and a desialylated andgalactosylated NNAS antibody.

FIG. 54 depicts the results of MALDI-TOF MS analysis to determine theglycan structures of a mouse NNAS antibody and a sialylated NNASantibody.

FIG. 55 depicts the characterization of Man-6-P receptor (CI-MPR) boundto bis Man-6-P glycan-conjugated polyclonal and monoclonal antibodiesthrough native Fc glycan or hinge disulfides using native PAGE.

FIG. 56 depicts the characterization of enzyme modified and glycopeptideconjugated NNAS antibodies by SDS-PAGE (4-12% NuPAGE; reducing andnon-reducing) and ECL lectin blotting (reducing).

FIG. 57 depicts the results of terminal galactose quantitation in anNNAS antibody, a desialylated/galactosylated NNAS antibody, and aconjugated NNAS antibody in mol galactose or mol glycopeptide per molantibody.

FIG. 58 depicts the examination of lactose maleimide that had beenmodified with alpha-2,3-sialyltransferase and eluted from QAEpurification columns with 20 mM NaCl. The resultant eluate wascharacterized using MALDI-TOF MS and Dionex HPLC.

FIGS. 59A-59B depict the characterization of rabbit polyclonal antibodyconjugated with sialyllactose maleimide (thiol reaction) using SDS-PAGEand Dionex HPLC (sialic acid quantitation).

FIGS. 60A-60D depict the characterization of lactose maleimidesialylated with alpha-2,6-sialyltransferase and purified using aQAE-sepharose column. Analysis using Dionex HPLC is shown for (FIG. 60A)a lactose standard; (FIG. 60B) an alpha-2,6-sialyllactose standard;(FIG. 60C) a lactose maleimide standard; and (FIG. 60D) a fraction ofalpha-2,6-sialyllactose maleimide eluted from a QAE-sepharose column.

FIG. 61 depicts the characterization of a fraction ofalpha-2,6-sialyllactose maleimide eluted from a QAE-sepharose columnusing MALDI-TOF MS.

FIGS. 62A-62B depict the characterization of a control antibody, analpha-2,3-sialyllactose glycan conjugated polyclonal antibody, and analpha-2,6-sialyllactose glycan conjugated polyclonal antibody throughSDS-PAGE and Dionex HPLC (graph of sialic acid analysis shown).

FIG. 63 depicts the characterization of control and enzyme modified(desialylated/galactosylated) NNAS mutant antibodies using SDS-PAGE andlectin blotting.

FIG. 64 depicts the characterization through reducing and non-reducingSDS-PAGE of the PEGylated control antibody and Gal NNAS with variousamounts of galactose oxidase.

FIG. 65 depicts the results of estimated PEGylation of an antibody heavychain from previous galactose oxidase titration using ProteinSimple.

FIG. 66 depicts the characterization through reducing and non-reducingSDS-PAGE of the PEGylated control antibody and Gal NNAS with variousmolar excess of PEG over antibody.

FIG. 67 depicts the results of estimated PEGylation of an antibody heavychain from previous PEG titration using ProteinSimple.

FIG. 68 is a structural drawing of aminooxy glycopeptide,lactose₃-Cys₃Gly₄.

FIGS. 69A-69B depict the characterization through reducing SDS-PAGE ofthe PEGylated control antibody and Gal NNAS with galactose oxidase inthe absence of copper acetate (FIG. 69A) and in the presence of varyingamounts of copper acetate (FIG. 69A and FIG. 69B).

FIG. 70 depicts the characterization of enzyme modified wild-type,A114N, NNAS, and A114N/NNAS Herceptin by SDS-PAGE (4-12% NuPAGE;reducing and non-reducing) and ECL lectin blotting (reducing) along withthe results of terminal galactose quantitation in mol galactose per molantibody.

FIG. 71 is a table depicting the sialic acid content (in mol/mol) ofwild-type and mutant antibodies as measured using Dionex HPLC.

FIG. 72 depicts the characterization of the PEGylation of wild-type andmutant antibodies through reducing and non-reducing SDS-PAGE.

FIG. 73 is a table depicting the PEGylation (in mol/mol) of wild-typeand mutant antibodies estimated using ProteinSimple.

FIG. 74 is a series of photos depicting immunofluorescence staining ofHepG2 cell uptake of control, enzyme modified (withgalactosyltransferase), or conjugated (with lactose aminoxy or lactosemaleimide) antibodies.

FIG. 75 is a depiction of an exemplary trivalent GalNAc glycan.

FIG. 76 depicts the results of surface plasmon resonance experimentsused to assess the binding of trivalent GalNAc glycan-conjugatedantibodies to ASGPR subunit H1.

FIG. 77 is a depiction of a trivalent GalNAc-containing glycan and atrivalent galactose-containing glycopeptide used for conjugation.

FIG. 78 depicts the results of surface plasmon resonance experimentsused to assess the binding of trivalent GalNAc-conjugated and trivalentgalactose containing glycopeptide-conjugated recombinant lysosomalenzymes to ASGPR subunit H1.

FIGS. 79A-79D are depiction of additional trivalent GalNAc glycans.

FIG. 80 is a depiction of the results of conjugation of periodateoxidized recombinant lysosomal enzyme rhGAA with an excess of trivalentGalNAc glycan C12 (at 20, 40, 80, and 200-fold molar excess glycan overrhGAA). The resulting glycan conjugated per rhGAA is depicted.

FIG. 81 depicts ASGPR binding of a recombinant lysosomal enzymesconjugated with trivalent GalNAc glycan C12 on Biacore. Enzymesconjugated with 20 (conjugate 1), 40, 80, and 200-fold (conjugate 4)excess of glycan all show strong binding to ASGPR subunit 1. There is nosignificant difference in binding among the conjugates (conjugates 1 to4).

DETAILED DESCRIPTION

The current disclosure provides binding polypeptides (e.g., antibodies),and effector moiety conjugates (e.g., targeting moiety conjugates)thereof. In certain embodiments, the conjugates comprise asite-specifically engineered drug-glycan linkage within native ormodified glycans of an antigen binding polypeptide such as an IgGmolecule. The current disclosure also provides nucleic acids encodingantigen-binding polypeptides, recombinant expression vectors and hostcells for making antigen-binding polypeptides. Methods of using theantigen-binding polypeptides disclosed herein to treat disease are alsoprovided.

I. Definitions

Unless otherwise defined herein, scientific and technical terms usedherein have the meanings that are commonly understood by those ofordinary skill in the art. In the event of any latent ambiguity,definitions provided herein take precedent over any dictionary orextrinsic definition. Unless otherwise required by context, singularterms shall include pluralities and plural terms shall include thesingular. The use of “or” means “and/or” unless stated otherwise. Theuse of the term “including”, as well as other forms, such as “includes”and “included”, is not limiting.

Generally, nomenclatures used in connection with cell and tissueculture, molecular biology, immunology, microbiology, genetics andprotein and nucleic acid chemistry and hybridization described hereinare those well-known and commonly used in the art. The methods andtechniques provided herein are generally performed according toconventional methods well known in the art and as described in variousgeneral and more specific references that are cited and discussedthroughout the present specification unless otherwise indicated.Enzymatic reactions and purification techniques are performed accordingto manufacturer's specifications, as commonly accomplished in the art oras described herein. The nomenclatures used in connection with, and thelaboratory procedures and techniques of, analytical chemistry, syntheticorganic chemistry, and medicinal and pharmaceutical chemistry describedherein are those well-known and commonly used in the art. Standardtechniques are used for chemical syntheses, chemical analyses,pharmaceutical preparation, formulation, and delivery, and treatment ofpatients.

That the disclosure may be more readily understood, select terms aredefined below.

The term “polypeptide” refer to any polymeric chain of amino acids andencompasses native or artificial proteins, polypeptide analogs orvariants of a protein sequence, or fragments thereof, unless otherwisecontradicted by context. A polypeptide may be monomeric or polymeric.For an antigenic polypeptide, a fragment of a polypeptide optionallycontains at least one contiguous or nonlinear epitope of a polypeptide.The precise boundaries of the at least one epitope fragment can beconfirmed using ordinary skill in the art. A polypeptide fragmentcomprises at least about 5 contiguous amino acids, at least about 10contiguous amino acids, at least about 15 contiguous amino acids, or atleast about 20 contiguous amino acids, for example.

The term “isolated protein” or “isolated polypeptide” refer to a proteinor polypeptide that by virtue of its origin or source of derivation isnot associated with naturally associated components that accompany it inits native state; is substantially free of other proteins from the samespecies; is expressed by a cell from a different species; or does notoccur in nature. Thus, a protein or polypeptide that is chemicallysynthesized or synthesized in a cellular system different from the cellfrom which it naturally originates will be “isolated” from its naturallyassociated components. A protein or polypeptide may also be renderedsubstantially free of naturally associated components by isolation usingprotein purification techniques well known in the art.

As used herein, the term “binding protein” or “binding polypeptide”shall refer to a protein or polypeptide (e.g., an antibody or fragmentthereof) that contains at least one binding site which is responsiblefor selectively binding to a target antigen of interest (e.g., a humanantigen). Exemplary binding sites include an antibody variable domain, aligand binding site of a receptor, or a receptor binding site of aligand. In certain aspects, the binding proteins or binding polypeptidescomprise multiple (e.g., two, three, four, or more) binding sites. Incertain aspects, the binding protein or binding polypeptide is not atherapeutic enzyme.

As used herein, the term “native residue” shall refer to an amino acidresidue that occurs naturally at a particular amino acid position of abinding polypeptide (e.g., an antibody or fragment thereof) and whichhas not been modified, introduced, or altered by the hand of man. Asused herein, the term “altered binding protein,” “altered bindingpolypeptide,” “modified binding protein” or “modified bindingpolypeptide” shall refer to binding polypeptides and/or binding proteins(e.g., an antibody or fragment thereof) comprising at least one aminoacid substitution, deletion and/or addition relative to the native(i.e., wild-type) amino acid sequence, and/or a mutation that results inaltered glycosylation (e.g., hyperglycosylation, hypoglycosylationand/or aglycosylation) at one or more amino acid positions relative tothe native (i.e., wild-type) amino acid sequence.

As used herein, the term “initial binding polypeptide” or “initialbinding protein” shall refer to a binding polypeptide or binding proteinthat is contacted with a mildly oxidizing agent to produce a “precursorbinding polypeptide” or a “precursor binding protein,” respectively (seeFIGS. 1A-C). As used herein, the term “precursor binding polypeptide” or“precursor binding protein” shall refer to a mildly oxidized polypeptideor protein that can be reacted with one or more of the effector moietiesdescribed herein. In certain embodiments, an initial bindingpolypeptide, initial binding protein, precursor binding polypeptide,and/or precursor binding protein (e.g., an antibody or fragment thereof)contains at least one binding site which is responsible for selectivelybinding to a target antigen of interest (e.g., a human antigen).Exemplary binding sites include an antibody variable domain, a ligandbinding site of a receptor, or a receptor binding site of a ligand. Incertain aspects, an initial binding polypeptide, initial bindingprotein, precursor binding polypeptide, and/or precursor binding proteincomprises multiple (e.g., two, three, four, or more) binding sites. Incertain aspects, the initial binding polypeptide, initial bindingprotein, precursor binding polypeptide, and/or precursor binding proteinis not a therapeutic enzyme. An initial binding polypeptide, initialbinding protein, precursor binding polypeptide, and/or precursor bindingprotein may have a wild-type sequence or they may comprise at least oneamino acid substitution, deletion and/or addition relative to the native(i.e., wild-type) amino acid sequence, and/or a mutation that results inaltered glycosylation (e.g., hyperglycosylation, hypoglycosylationand/or aglycosylation) at one or more amino acid positions relative tothe native (i.e., wild-type) amino acid sequence.

The term “ligand” refers to any substance capable of binding, or ofbeing bound, to another substance. Similarly, the term “antigen” refersto any substance to which an antibody may be generated. Although“antigen” is commonly used in reference to an antibody bindingsubstrate, and “ligand” is often used when referring to receptor bindingsubstrates, these terms are not distinguishing, one from the other, andencompass a wide range of overlapping chemical entities. For theavoidance of doubt, antigen and ligand are used interchangeablythroughout herein. Antigens/ligands may be a peptide, a polypeptide, aprotein, an aptamer, a polysaccharide, a sugar molecule, a carbohydrate,a lipid, an oligonucleotide, a polynucleotide, a synthetic molecule, aninorganic molecule, an organic molecule, and any combination thereof.

The term “specifically binds” as used herein, refers to the ability ofan antibody or an antigen-binding fragment thereof to bind to an antigenwith a dissociation constant (Kd) of at most about 1×10⁻⁶ M, 1×10⁻⁷ M,1×10⁻⁸ M, 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, 1×10⁻¹² M, or less, and/or tobind to an antigen with an affinity that is at least two-fold greaterthan its affinity for a nonspecific antigen.

As used herein, the term “antibody” refers to such assemblies (e.g.,intact antibody molecules, antibody fragments, or variants thereof)which have significant known specific immunoreactive activity to anantigen of interest (e.g. a tumor associated antigen). Antibodies andimmunoglobulins comprise light and heavy chains, with or without aninterchain covalent linkage between them. Basic immunoglobulinstructures in vertebrate systems are relatively well understood.

As will be discussed in more detail below, the generic term “antibody”comprises five distinct classes of antibody that can be distinguishedbiochemically. While all five classes of antibodies are clearly withinthe scope of the current disclosure, the following discussion willgenerally be directed to the IgG class of immunoglobulin molecules. Withregard to IgG, immunoglobulins comprise two identical light chains ofmolecular weight approximately 23,000 Daltons, and two identical heavychains of molecular weight 53,000-70,000. The four chains are joined bydisulfide bonds in a “Y” configuration wherein the light chains bracketthe heavy chains starting at the mouth of the “Y” and continuing throughthe variable region.

Light chains of immunoglobulin are classified as either kappa or lambda(κ,λ). Each heavy chain class may be bound with either a kappa or lambdalight chain. In general, the light and heavy chains are covalentlybonded to each other, and the “tail” portions of the two heavy chainsare bonded to each other by covalent disulfide linkages or non-covalentlinkages when the immunoglobulins are generated either by hybridomas, Bcells, or genetically engineered host cells. In the heavy chain, theamino acid sequences run from an N-terminus at the forked ends of the Yconfiguration to the C-terminus at the bottom of each chain. Thoseskilled in the art will appreciate that heavy chains are classified asgamma, mu, alpha, delta, or epsilon, (γ, μ, α, δ, ε) with somesubclasses among them (e.g., γ1-γ4). It is the nature of this chain thatdetermines the “class” of the antibody as IgG, IgM, IgA IgG, or IgE,respectively. The immunoglobulin isotype subclasses (e.g., IgG1, IgG2,IgG3, IgG4, IgA1, etc.) are well characterized and are known to conferfunctional specialization. Modified versions of each of these classesand isotypes are readily discernable to the skilled artisan in view ofthe instant disclosure and, accordingly, are within the scope of thecurrent disclosure.

Both the light and heavy chains are divided into regions of structuraland functional homology. The term “region” refers to a part or portionof an immunoglobulin or antibody chain and includes constant region orvariable regions, as well as more discrete parts or portions of saidregions. For example, light chain variable regions include“complementarity determining regions” or “CDRs” interspersed among“framework regions” or “FRs”, as defined herein.

The regions of an immunoglobulin heavy or light chain may be defined as“constant” (C) region or “variable” (V) regions, based on the relativelack of sequence variation within the regions of various class membersin the case of a “constant region”, or the significant variation withinthe regions of various class members in the case of a “variableregions”. The terms “constant region” and “variable region” may also beused functionally. In this regard, it will be appreciated that thevariable regions of an immunoglobulin or antibody determine antigenrecognition and specificity. Conversely, the constant regions of animmunoglobulin or antibody confer important effector functions such assecretion, transplacental mobility, Fc receptor binding, complementbinding, and the like. The subunit structures and three dimensionalconfigurations of the constant regions of the various immunoglobulinclasses are well known.

The constant and variable regions of immunoglobulin heavy and lightchains are folded into domains. The term “domain” refers to a globularregion of a heavy or light chain comprising peptide loops (e.g.,comprising 3 to 4 peptide loops) stabilized, for example, by (3-pleatedsheet and/or intrachain disulfide bond. Constant region domains on thelight chain of an immunoglobulin are referred to interchangeably as“light chain constant region domains”, “CL regions” or “CL domains”.Constant domains on the heavy chain (e.g. hinge, CH1, CH2 or CH3domains) are referred to interchangeably as “heavy chain constant regiondomains”, “CH” region domains or “CH domains”. Variable domains on thelight chain are referred to interchangeably as “light chain variableregion domains”, “VL region domains or “VL domains”. Variable domains onthe heavy chain are referred to interchangeably as “heavy chain variableregion domains”, “VH region domains” or “VH domains”.

By convention the numbering of the variable constant region domainsincreases as they become more distal from the antigen binding site oramino-terminus of the immunoglobulin or antibody. The N-terminus of eachheavy and light immunoglobulin chain is a variable region and at theC-terminus is a constant region; the CH3 and CL domains actuallycomprise the carboxy-terminus of the heavy and light chain,respectively. Accordingly, the domains of a light chain immunoglobulinare arranged in a VL-CL orientation, while the domains of the heavychain are arranged in the VH-CH1-hinge-CH2-CH3 orientation.

Amino acid positions in a heavy chain constant region, including aminoacid positions in the CH1, hinge, CH2, CH3, and CL domains, may benumbered according to the Kabat index numbering system (see Kabat et al,in “Sequences of Proteins of Immunological Interest”, U.S. Dept. Healthand Human Services, 5th edition, 1991). Alternatively, antibody aminoacid positions may be numbered according to the EU index numberingsystem (see Kabat et al, ibid).

As used herein, the term “VH domain” includes the amino terminalvariable domain of an immunoglobulin heavy chain, and the term “VLdomain” includes the amino terminal variable domain of an immunoglobulinlight chain.

As used herein, the term “CH1 domain” includes the first (most aminoterminal) constant region domain of an immunoglobulin heavy chain thatextends, e.g., from about positions 114-223 in the Kabat numberingsystem (EU positions 118-215). The CH1 domain is adjacent to the VHdomain and amino terminal to the hinge region of an immunoglobulin heavychain molecule, and does not form a part of the Fc region of animmunoglobulin heavy chain.

As used herein, the term “hinge region” includes the portion of a heavychain molecule that joins the CH1 domain to the CH2 domain. This hingeregion comprises approximately 25 residues and is flexible, thusallowing the two N-terminal antigen binding regions to moveindependently. Hinge regions can be subdivided into three distinctdomains: upper, middle, and lower hinge domains (Roux et al. J. Immunol.1998, 161:4083).

As used herein, the term “CH2 domain” includes the portion of a heavychain immunoglobulin molecule that extends, e.g., from about positions244-360 in the Kabat numbering system (EU positions 231-340). The CH2domain is unique in that it is not closely paired with another domain.Rather, two N-linked branched carbohydrate chains are interposed betweenthe two CH2 domains of an intact native IgG molecule. In one embodiment,a binding polypeptide of the current disclosure comprises a CH2 domainderived from an IgG1 molecule (e.g. a human IgG1 molecule).

As used herein, the term “CH3 domain” includes the portion of a heavychain immunoglobulin molecule that extends approximately 110 residuesfrom N-terminus of the CH2 domain, e.g., from about positions 361-476 ofthe Kabat numbering system (EU positions 341-445). The CH3 domaintypically forms the C-terminal portion of the antibody. In someimmunoglobulins, however, additional domains may extend from CH3 domainto form the C-terminal portion of the molecule (e.g. the CH4 domain inthe μ chain of IgM and the e chain of IgE). In one embodiment, a bindingpolypeptide of the current disclosure comprises a CH3 domain derivedfrom an IgG1 molecule (e.g. a human IgG1 molecule).

As used herein, the term “CL domain” includes the constant region domainof an immunoglobulin light chain that extends, e.g., from about Kabatposition 107A-216. The CL domain is adjacent to the VL domain. In oneembodiment, a binding polypeptide of the current disclosure comprises aCL domain derived from a kappa light chain (e.g., a human kappa lightchain).

As used herein, the term “Fc region” is defined as the portion of aheavy chain constant region beginning in the hinge region just upstreamof the papain cleavage site (i.e. residue 216 in IgG, taking the firstresidue of heavy chain constant region to be 114) and ending at theC-terminus of the antibody. Accordingly, a complete Fc region comprisesat least a hinge domain, a CH2 domain, and a CH3 domain.

The term “native Fc” as used herein refers to a molecule comprising thesequence of a non-antigen-binding fragment resulting from digestion ofan antibody or produced by other means, whether in monomeric ormultimeric form, and can contain the hinge region. The originalimmunoglobulin source of the native Fc is typically of human origin andcan be any of the immunoglobulins, such as IgG1 and IgG2. Native Fcmolecules are made up of monomeric polypeptides that can be linked intodimeric or multimeric forms by covalent (i.e., disulfide bonds) andnon-covalent association. The number of intermolecular disulfide bondsbetween monomeric subunits of native Fc molecules ranges from 1 to 4depending on class (e.g., IgG, IgA, and IgE) or subclass (e.g., IgG1,IgG2, IgG3, IgA1, and IgGA2). One example of a native Fc is adisulfide-bonded dimer resulting from papain digestion of an IgG. Theterm “native Fc” as used herein is generic to the monomeric, dimeric,and multimeric forms.

The term “Fc variant” as used herein refers to a molecule or sequencethat is modified from a native Fc but still comprises a binding site forthe salvage receptor, FcRn (neonatal Fc receptor). Exemplary Fcvariants, and their interaction with the salvage receptor, are known inthe art. Thus, the term “Fc variant” can comprise a molecule or sequencethat is humanized from a non-human native Fc. Furthermore, a native Fccomprises regions that can be removed because they provide structuralfeatures or biological activity that are not required for theantibody-like binding polypeptides featured in the invention. Thus, theterm “Fc variant” comprises a molecule or sequence that lacks one ormore native Fc sites or residues, or in which one or more Fc sites orresidues has be modified, that affect or are involved in: (1) disulfidebond formation, (2) incompatibility with a selected host cell, (3)N-terminal heterogeneity upon expression in a selected host cell, (4)glycosylation, (5) interaction with complement, (6) binding to an Fcreceptor other than a salvage receptor, or (7) antibody-dependentcellular cytotoxicity (ADCC).

The term “Fc domain” as used herein encompasses native Fc and Fcvariants and sequences as defined above. As with Fc variants and nativeFc molecules, the term “Fc domain” includes molecules in monomeric ormultimeric form, whether digested from whole antibody or produced byother means.

As indicated above, the variable regions of an antibody allow it toselectively recognize and specifically bind epitopes on antigens. Thatis, the VL domain and VH domain of an antibody combine to form thevariable region (Fv) that defines a three dimensional antigen bindingsite. This quaternary antibody structure forms the antigen binding sitepresent at the end of each arm of the Y. More specifically, the antigenbinding site is defined by three complementary determining regions(CDRs) on each of the heavy and light chain variable regions. As usedherein, the term “antigen binding site” includes a site thatspecifically binds (immunoreacts with) an antigen (e.g., a cell surfaceor soluble antigen). The antigen binding site includes an immunoglobulinheavy chain and light chain variable region and the binding site formedby these variable regions determines the specificity of the antibody. Anantigen binding site is formed by variable regions that vary from oneantibody to another. The altered antibodies of the current disclosurecomprise at least one antigen binding site.

In certain embodiments, binding polypeptides of the current disclosurecomprise at least two antigen binding domains that provide for theassociation of the binding polypeptide with the selected antigen. Theantigen binding domains need not be derived from the same immunoglobulinmolecule. In this regard, the variable region may or be derived from anytype of animal that can be induced to mount a humoral response andgenerate immunoglobulins against the desired antigen. As such, thevariable region of the a binding polypeptide may be, for example, ofmammalian origin e.g., may be human, murine, rat, goat, sheep, non-humanprimate (such as cynomolgus monkeys, macaques, etc.), lupine, or camelid(e.g., from camels, llamas and related species).

In naturally occurring antibodies, the six CDRs present on eachmonomeric antibody are short, non-contiguous sequences of amino acidsthat are specifically positioned to form the antigen binding site as theantibody assumes its three dimensional configuration in an aqueousenvironment. The remainder of the heavy and light variable domains showless inter-molecular variability in amino acid sequence and are termedthe framework regions. The framework regions largely adopt a β-sheetconformation and the CDRs form loops which connect, and in some casesform part of, the β-sheet structure. Thus, these framework regions actto form a scaffold that provides for positioning the six CDRs in correctorientation by inter-chain, non-covalent interactions. The antigenbinding domain formed by the positioned CDRs defines a surfacecomplementary to the epitope on the immunoreactive antigen. Thiscomplementary surface promotes the non-covalent binding of the antibodyto the immunoreactive antigen epitope.

Exemplary binding polypeptides include antibody variants. As usedherein, the term “antibody variant” includes synthetic and engineeredforms of antibodies which are altered such that they are not naturallyoccurring, e.g., antibodies that comprise at least two heavy chainportions but not two complete heavy chains (such as, domain deletedantibodies or minibodies); multispecific forms of antibodies (e.g.,bispecific, trispecific, etc.) altered to bind to two or more differentantigens or to different epitopes on a single antigen); heavy chainmolecules joined to scFv molecules and the like. In addition, the term“antibody variant” includes multivalent forms of antibodies (e.g.,trivalent, tetravalent, etc., antibodies that bind to three, four ormore copies of the same antigen.

As used herein the term “valency” refers to the number of potentialtarget binding sites in a polypeptide. Each target binding sitespecifically binds one target molecule or specific site on a targetmolecule. When a polypeptide comprises more than one target bindingsite, each target binding site may specifically bind the same ordifferent molecules (e.g., may bind to different ligands or differentantigens, or different epitopes on the same antigen). The subjectbinding polypeptides typically has at least one binding site specificfor a human antigen molecule.

The term “specificity” refers to the ability to specifically bind (e.g.,immunoreact with) a given target antigen (e.g., a human target antigen).A binding polypeptide may be monospecific and contain one or morebinding sites which specifically bind a target or a polypeptide may bemultispecific and contain two or more binding sites which specificallybind the same or different targets. In certain embodiments, a bindingpolypeptide is specific for two different (e.g., non-overlapping)portions of the same target. In certain embodiments, a bindingpolypeptide is specific for more than one target. Exemplary bindingpolypeptides (e.g., antibodies) which comprise antigen binding sitesthat bind to antigens expressed on tumor cells are known in the art andone or more CDRs from such antibodies can be included in an antibodyfeatured in the invention.

The term “linking moiety” includes moieties which are capable of linkingthe effector moiety to the binding polypeptides disclosed herein. Thelinking moiety may be selected such that it is cleavable (e.g.,enzymatically cleavable or pH-sensitive) or non-cleavable. Exemplarylinking moieties are set forth in Table 2 herein.

As used herein, the term “effector moiety” comprises agents (e.g.proteins, nucleic acids, lipids, carbohydrates, glycopeptides, andfragments thereof) with biological or other functional activity. Forexample, a modified binding polypeptide comprising an effector moietyconjugated to a binding polypeptide has at least one additional functionor property as compared to the unconjugated antibody. For example, theconjugation of a cytotoxic drug (e.g., an effector moiety) to bindingpolypeptide results in the formation of a binding polypeptide with drugcytotoxicity as second function (i.e. in addition to antigen binding).In another example, the conjugation of a second binding polypeptide tothe binding polypeptide may confer additional binding properties. Incertain embodiments, where the effector moiety is a genetically encodedtherapeutic or diagnostic protein or nucleic acid, the effector moietymay be synthesized or expressed by either peptide synthesis orrecombinant DNA methods that are well known in the art. In anotheraspect, where the effector moiety is a non-genetically encoded peptide,or a drug moiety, the effector moiety may be synthesized artificially orpurified from a natural source. As used herein, the term “drug moiety”includes anti-inflammatory, anticancer, anti-infective (e.g.,anti-fungal, antibacterial, anti-parasitic, anti-viral, etc.), andanesthetic therapeutic agents. In a further embodiment, the drug moietyis an anticancer or cytotoxic agent. Compatible drug moieties may alsocomprise prodrugs. Exemplary effector moieties are set forth in Table 1herein.

As used herein the term “mildly oxidizing” refers to a reagent thataccepts an electron from another species (and is therefore itselfreduced), but does not attract those electrons very strongly. Examplesof mildly oxidizing agents include, but are not limited to, periodateoxidase and galactose oxidase.

In certain embodiments, an “effector moiety” comprises a “targetingmoiety.” As used herein, the term “targeting moiety” refers to aneffector moiety that binds to a target molecule. Targeting moieties cancomprise, without limitation, proteins, nucleic acids, lipids,carbohydrates (e.g., glycans), and combinations thereof (e.g.,glycoproteins, glycopeptides, and glycolipids).

As used herein, the term “prodrug” refers to a precursor or derivativeform of a pharmaceutically active agent that is less active, reactive orprone to side effects as compared to the parent drug and is capable ofbeing enzymatically activated or otherwise converted into a more activeform in vivo. Prodrugs compatible with the compositions of the currentdisclosure include, but are not limited to, phosphate-containingprodrugs, amino acid-containing prodrugs, thiophosphate-containingprodrugs, sulfate-containing prodrugs, peptide-containing prodrugs,β-lactam-containing prodrugs, optionally substitutedphenoxyacetamide-containing prodrugs or optionally substitutedphenylacetamide-containing prodrugs, 5-fluorocytosine and other5-fluorouridine prodrugs that can be converted to the more activecytotoxic free drug. One skilled in the art may make chemicalmodifications to the desired drug moiety or its prodrug in order to makereactions of that compound more convenient for purposes of preparingmodified binding polypeptides of the current disclosure. The drugmoieties also include derivatives, pharmaceutically acceptable salts,esters, amides, and ethers of the drug moieties described herein.Derivatives include modifications to drugs identified herein which mayimprove or not significantly reduce a particular drug's desiredtherapeutic activity.

As used herein, the term “anticancer agent” includes agents which aredetrimental to the growth and/or proliferation of neoplastic or tumorcells and may act to reduce, inhibit or destroy malignancy. Examples ofsuch agents include, but are not limited to, cytostatic agents,alkylating agents, antibiotics, cytotoxic nucleosides, tubulin bindingagents, hormones, hormone antagonists, cytotoxic agents, and the like.Cytotoxic agents include tomaymycin derivatives, maytansine derivatives,cryptophycine derivatives, anthracycline derivatives, bisphosphonatederivatives, leptomycin derivatives, streptonigrin derivatives,auristatine derivatives, and duocarmycin derivatives. Any agent thatacts to retard or slow the growth of immunoreactive cells or malignantcells is within the scope of the current disclosure.

The term “antigen” or “target antigen” as used herein refers to amolecule or a portion of a molecule that is capable of being bound bythe binding site of a binding polypeptide. A target antigen may have oneor more epitopes.

The term “salt” comprises a metal ion. For example, a metal ion includesbut is not limited to an alkali metal (Group Ia), e.g. lithium, sodium,and potassium, an alkaline earth metal (Group IIa), e.g., magnesium andcalcium, a transition metal, e.g., copper, zinc, nickel, iron, andmanganese in usual valences. Exemplary usual valences of metals include,for example, sodium(I), calcium(II), magnesium(II), zinc(II), copper(I),and copper(II). Example salts comprising a metal ion, include but arenot limited to, copper(II) acetate (Cu₂(OAc)₄), zinc(II) acetate(Zn₂(OAc)₄), iron chloride (Fe₃Cl₃) and calcium chloride (CaCl₂)).

II. Binding Polypeptides

In one aspect, the current disclosure provides binding polypeptides(e.g., antibodies, antibody fragments, antibody variants, and fusionproteins) comprising a glycosylated domain, e.g., a glycosylatedconstant domain. The binding polypeptides disclosed herein encompass anybinding polypeptide that comprises a domain having an N-linkedglycosylation site. In certain embodiments, the binding polypeptide isan antibody, or fragment or derivative thereof Δny antibody from anysource or species can be employed in the binding polypeptides disclosedherein. Suitable antibodies include without limitation, humanantibodies, humanized antibodies or chimeric antibodies.

In certain embodiments, the glycosylated domain is an Fc domain. Incertain embodiments, the glycosylation domain is a native glycosylationdomain at N297, according to EU numbering.

In other embodiments, the glycosylation domain is an engineeredglycosylation domain. Exemplary engineered glycosylation domains in Fcdomain comprise an asparagine residue at amino acid position 298,according to EU numbering, and/or a serine or threonine residue at aminoacid position 300, according to EU numbering.

Fc domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA andIgE) and species can be used in the binding polypeptides disclosedherein. Chimeric Fc domains comprising portions of Fc domains fromdifferent species or Ig classes can also be employed. In certainembodiments, the Fc domain is a human IgG1 Fc domain. In the case of ahuman IgG1 Fc domain, mutation of the wild-type amino acid at Kabatposition 298 to an asparagine and Kabat position 300 to a serine orthreonine results in the formation of an N-linked glycosylationconsensus site (i.e., the N-X-T/S sequon, where X is any amino acidexcept proline). However, in the case of Fc domains of other speciesand/or Ig classes or isotypes, the skill artisan will appreciate that itmay be necessary to mutate Kabat position 299 of the Fc domain if aproline residue is present to recreate an N-X-T/S sequon.

In other embodiments, the current disclosure provides bindingpolypeptides (e.g., antibodies, antibody fragments, antibody variants,and fusion proteins) comprising at least one CH1 domain having anN-linked glycosylation site. Such exemplary binding polypeptides maycomprise, for example, and engineered glycosylation site at position114, according to Kabat numbering.

CH1 domains from any immunoglobulin class (e.g., IgM, IgG, IgD, IgA andIgE) and species can be used in the binding polypeptides disclosedherein. Chimeric CH1 domains comprising portions of CHI domains fromdifferent species or Ig classes can also be employed. In certainembodiments, the CH1 domain is a human IgG1 CH1 domain. In the case of ahuman IgG1 domain, mutation of the wild-type amino acid at position 114to an asparagine results in the formation of an N-linked glycosylationconsensus site (i.e., the N-X-T/S sequon, where X is any amino acidexcept proline). However, in the case of other CH1 domains of otherspecies and/or Ig classes or isotypes, the skilled artisan willappreciate that it may be necessary to mutate positions 115 and/or 116of the CH1 domain to create an N-X-T/S sequon.

In certain embodiments, the binding polypeptide of the currentdisclosure may comprise an antigen binding fragment of an antibody. Theterm “antigen-binding fragment” refers to a polypeptide fragment of animmunoglobulin or antibody which binds antigen or competes with intactantibody (i.e., with the intact antibody from which they were derived)for antigen binding (i.e., specific binding). Antigen binding fragmentscan be produced by recombinant or biochemical methods that are wellknown in the art. Exemplary antigen-binding fragments include Fv, Fab,Fab′, and (Fab′)2. In exemplary embodiments, the antigen-bindingfragment of the current disclosure is an altered antigen-bindingfragment comprising at least one engineered glycosylation site. In oneexemplary embodiment, an altered antigen binding fragment of the currentdisclosure comprises an altered VH domain described supra. In anotherexemplary embodiment, an altered antigen binding fragment of the currentdisclosure comprises an altered CH1 domain described.

In exemplary embodiments, the binding polypeptide comprises a singlechain variable region sequence (ScFv). Single chain variable regionsequences comprise a single polypeptide having one or more antigenbinding sites, e.g., a VL domain linked by a flexible linker to a VHdomain. ScFv molecules can be constructed in a VH-linker-VL orientationor VL-linker-VH orientation. The flexible hinge that links the VL and VHdomains that make up the antigen binding site includes from about 10 toabout 50 amino acid residues. Connecting peptides are known in the art.Binding polypeptides may comprise at least one scFv and/or at least oneconstant region. In one embodiment, a binding polypeptide of the currentdisclosure may comprise at least one scFv linked or fused to an antibodyor fragment comprising a CH1 domain (e.g. a CH1 domain comprising anasparagine residue at Kabat position 114/EU position 118) and/or a CH2domain (e.g. a CH2 domain comprising an asparagine residue at EUposition 298, and a serine or threonine residue at EU position 300).

In certain exemplary embodiments, a binding polypeptide of the currentdisclosure is a multivalent (e.g., tetravalent) antibody which isproduced by fusing a DNA sequence encoding an antibody with a ScFvmolecule (e.g., an altered ScFv molecule). For example, in oneembodiment, these sequences are combined such that the ScFv molecule(e.g., an altered ScFv molecule) is linked at its N-terminus orC-terminus to an Fc fragment of an antibody via a flexible linker (e.g.,a gly/ser linker). In another embodiment a tetravalent antibody of thecurrent disclosure can be made by fusing an ScFv molecule to aconnecting peptide, which is fused to a CH1 domain (e.g. a CH1 domaincomprising an asparagine residue at Kabat position 114/EU position 118)to construct an ScFv-Fab tetravalent molecule.

In another embodiment, a binding polypeptide of the current disclosureis an altered minibody. An altered minibody of the current disclosure isa dimeric molecule made up of two polypeptide chains each comprising anScFv molecule (e.g., an altered ScFv molecule comprising an altered VHdomain described supra) which is fused to a CH3 domain or portionthereof via a connecting peptide. Minibodies can be made by constructingan ScFv component and connecting peptide-CH3 components using methodsdescribed in the art (see, e.g., U.S. Pat. No. 5,837,821 or WO94/09817A1). In another embodiment, a tetravalent minibody can beconstructed. Tetravalent minibodies can be constructed in the samemanner as minibodies, except that two ScFv molecules are linked using aflexible linker. The linked scFv-scFv construct is then joined to a CH3domain.

In another embodiment, a binding polypeptide of the current disclosurecomprises a diabody. Diabodies are dimeric, tetravalent molecules eachhaving a polypeptide similar to scFv molecules, but usually having ashort (less than 10, e.g., 1-5) amino acid residue linker connectingboth variable domains, such that the VL and VH domains on the samepolypeptide chain cannot interact. Instead, the VL and VH domain of onepolypeptide chain interact with the VH and VL domain (respectively) on asecond polypeptide chain (see, for example, WO 02/02781). Diabodies ofthe current disclosure comprise an scFv molecule fused to a CH3 domain.

In other embodiments, the binding polypeptides comprise multispecific ormultivalent antibodies comprising one or more variable domain in serieson the same polypeptide chain, e.g., tandem variable domain (TVD)polypeptides. Exemplary TVD polypeptides include the “double head” or“Dual-Fv” configuration described in U.S. Pat. No. 5,989,830. In theDual-Fv configuration, the variable domains of two different antibodiesare expressed in a tandem orientation on two separate chains (one heavychain and one light chain), wherein one polypeptide chain has two VHdomains in series separated by a peptide linker (VH1-linker-VH2) and theother polypeptide chain consists of complementary VL domains connectedin series by a peptide linker (VL1-linker-VL2). In the cross-over doublehead configuration, the variable domains of two different antibodies areexpressed in a tandem orientation on two separate polypeptide chains(one heavy chain and one light chain), wherein one polypeptide chain hastwo VH domains in series separated by a peptide linker (VH1-linker-VH2)and the other polypeptide chain consists of complementary VL domainsconnected in series by a peptide linker in the opposite orientation(VL2-linker-VL1). Additional antibody variants based on the “Dual-Fv”format include the Dual-Variable-Domain IgG (DVD-IgG) bispecificantibody (see U.S. Pat. No. 7,612,181 and the TBTI format (see US2010/0226923 A1). The addition of constant domains to respective chainsof the Dual-Fv (CH1-Fc to the heavy chain and kappa or lambda constantdomain to the light chain) leads to functional bispecific antibodieswithout any need for additional modifications (i.e., obvious addition ofconstant domains to enhance stability).

In another exemplary embodiment, the binding polypeptide comprises across-over dual variable domain IgG (CODV-IgG) bispecific antibody basedon a “double head” configuration (see US20120251541 A1, which isincorporated by reference herein in its entirety). CODV-IgG antibodyvariants have one polypeptide chain with VL domains connected in seriesto a CL domain (VL1-L1-VL2-L2-CL) and a second polypeptide chain withcomplementary VH domains connected in series in the opposite orientationto a CH1 domain (VH2-L3-VH1-L4-CH1), where the polypeptide chains form across-over light chain-heavy chain pair. In certain embodiment, thesecond polypeptide may be further connected to an Fc domain(VH2-L3-VH1-L4-CH1-Fc). In certain embodiments, linker L3 is at leasttwice the length of linker L1 and/or linker L4 is at least twice thelength of linker L2. For example, L1 and L2 may be 1-3 amino acidresidues in length, L3 may be 2 to 6 amino acid residues in length, andL4 may be 4 to 7 amino acid residues in length. Examples of suitablelinkers include a single glycine (Gly) residue; a diglycine peptide(Gly-Gly); a tripeptide (Gly-Gly-Gly); a peptide with four glycineresidues (Gly-Gly-Gly-Gly) (SEQ ID NO: 40); a peptide with five glycineresidues (Gly-Gly-Gly-Gly-Gly) (SEQ ID NO: 41); a peptide with sixglycine residues (Gly-Gly-Gly-Gly-Gly-Gly) (SEQ ID NO: 42); a peptidewith seven glycine residues (Gly-Gly-Gly-Gly-Gly-Gly-Gly) (SEQ ID NO:43); a peptide with eight glycine residues(Gly-Gly-Gly-Gly-Gly-Gly-Gly-Gly) (SEQ ID NO: 44). Other combinations ofamino acid residues may be used such as the peptide Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 45) and the peptide Gly-Gly-Gly-Gly-Ser-Gly-Gly-Gly-Gly-Ser(SEQ ID NO: 46).

In certain embodiments, the binding polypeptide comprises animmunoadhesin molecule comprising a non-antibody binding region (e.g., areceptor, ligand, or cell-adhesion molecule) fused to an antibodyconstant region (see e.g., Ashkenazi et al., Methods, 1995 8(2),104-115, which is incorporated by reference herein in its entirety)

In certain embodiments, the binding polypeptide comprisesimmunoglobulin-like domains. Suitable immunoglobulin-like domainsinclude, without limitation, fibronectin domains (see, for example,Koide et al. (2007), Methods Mol. Biol. 352: 95-109, which isincorporated by reference herein in its entirety), DARPin (see, forexample, Stumpp et al. (2008) Drug Discov. Today 13 (15-16): 695-701,which is incorporated by reference herein in its entirety), Z domains ofprotein A (see, Nygren et al. (2008) FEBS J. 275 (11): 2668-76, which isincorporated by reference herein in its entirety), Lipocalins (see, forexample, Skerra et al. (2008) FEBS J. 275 (11): 2677-83, which isincorporated by reference herein in its entirety), Affilins (see, forexample, Ebersbach et al. (2007) J Mol. Biol. 372 (1): 172-85, which isincorporated by reference herein in its entirety), Affitins (see, forexample, Krehenbrink et al. (2008). J Mol. Biol. 383 (5): 1058-68, whichis incorporated by reference herein in its entirety), Avimers (see, forexample, Silverman et al. (2005) Nat. Biotechnol. 23 (12): 1556-61,which is incorporated by reference herein in its entirety), Fynomers,(see, for example, Grabulovski et al. (2007) J Biol Chem 282 (5):3196-3204, which is incorporated by reference herein in its entirety),and Kunitz domain peptides (see, for example, Nixon et al. (2006) CurrOpin Drug Discov Devel 9 (2): 261-8, which is incorporated by referenceherein in its entirety).

III. N-Linked Glycans

In certain embodiments, the binding polypeptides featured in theinvention employ glycans that are “N-linked” via an asparagine residueto a glycosylation site in the polypeptide backbone of the bindingpolypeptide. The glycosylation site may be a native or engineeredglycosylation site. Additionally or alternatively, the glycan may be anative glycan or an engineered glycan (e.g., a modified glycan)containing one or more non-native linkages. “N-glycans” or “N-linkedglycans” are attached at an amide nitrogen of an asparagine or anarginine residue in a protein via an N-acetylglucosamine residue. These“N-linked glycosylation sites” occur in the peptide primary structurecontaining, for example, the amino acid sequenceasparagine-X-serine/threonine, where X is any amino acid residue exceptproline and aspartic acid. Such N-Glycans are fully described in, forexample, Drickamer K, Taylor M E (2006). Introduction to Glycobiology,2nd ed., which is incorporated herein by reference in its entirety.

In certain embodiments, glycoengineered binding proteins and/or bindingpolypeptides and methods of making glycoengineered binding proteinsand/or binding polypeptides are provided. As used herein, the term“glycoengineering” refers to any art-recognized method for altering theglycoform profile of a binding protein composition to generate a“modified glycan.”

As used herein the terms “G0 glycoform,” “G1 glycoform,” and “G2glycoform” refer to N-Glycan glycoforms that have zero, one or twoterminal galactose residues respectively. These terms include G0, G1,and G2 glycoforms that are fucosylated or comprise a bisectingN-acetylglucosamine residue.

In certain embodiments, the G1 and G2 glycoforms further comprise sialicacid residues linked to one or both of the terminal galactose residuesto form G151, G2S1 and G2S2 glycoforms. As used herein the terms “G1S1glycoform,” “G2S1 glycoform,” and “G252 glycoform” refer to N-Glycanglycoforms that have a sialic acid residue linked to the sole terminalgalactose residue in a G1 glycoform, one of the terminal galactoseresidue in a G2 glycoform, or both of the terminal galactose residues ina G2 glycoform, respectively. These terms include G1S1, G2S1 and G2S2glycoforms that are fucosylated or comprise a bisectingN-acetylglucosamine residue. In certain embodiments, the sialic acidresidues of G1S1, G2S1 and G2S2 glycoforms are linked byalpha-2,6-sialic acid linkages to the terminal galactose residue of eachglycoform in order to enhance the anti-inflammatory activity of thebinding molecule (see e.g., Anthony et al., PNAS 105: 19571-19578,2008).

As used herein the terms “G1F glycoform,” “G2F glycoform,” “G1S1Fglycoform,” “G2S1F glycoform,” and “G2S2F glycoform” refer to “G1glycoform,” “G2 glycoform” “G1S1 glycoform,” “G2S1 glycoform,” and “G2S2glycoform” that are fucosylated, respectively.

In certain exemplary embodiments, the binding polypeptide comprises thenative glycosylation site of an antibody Fc domain. This nativeglycosylation site comprises a wild-type asparagine residue at position297 of the Fc domain (N297), according to EU numbering. The nativeN-linked glycan that resides at this position is generally linkedthrough a (3-glycosylamine linkage to the nitrogen group of the N297side chain. However, other suitable art recognized linkages can also beemployed. An N297 N-linked glycan may contain a terminal mannose,N-acetyl-glucosamine, galactose or sialic acid.

In other exemplary embodiments, the binding polypeptides comprise one ormore engineered glycosylation sites. Such engineered glycosylation sitescomprise the substitution of one or more wild-type amino acids in thepolypeptide backbone of the binding polypeptide with an asparagineresidue that is capable of being N-glycosylated by the glycosylationenzymes of a cell. Exemplary engineered glycosylation sites include theintroduction of asparagine mutation at amino acid position 298 of the Fcdomain (298N) according to EU numbering or amino acid position 114 of aCH1 domain (114N) according to Kabat numbering (position 118 of a CH1domain according to EU numbering).

Any type of naturally occurring or synthetic (i.e., non-natural ormodified) N-linked glycan can be linked to a glycosylation site of abinding polypeptide featured in the invention. In certain embodiments,the glycan comprises a saccharide (e.g., a saccharide residue located atterminus of an oligosaccharide) that can be oxidized (e.g., by periodatetreatment or galactose oxidase) to produce a group suitable forconjugation to an effector moiety (e.g., a reactive aldehyde group).Suitable oxidizable saccharides include, without limitation, galactoseand sialic acid (e.g., N-Acetylneuraminic acid). In certain embodiments,the glycan is a biantennary glycan. In certain embodiments, the glycanis a naturally occurring mammalian glycoform.

Glycosylation can be achieved through any means known in the art. Incertain embodiments, the glycosylation is achieved by expression of thebinding polypeptides in cells capable of N-linked glycosylation. Anynatural or engineered cell (e.g., prokaryotic or eukaryotic (e.g., CHOor NS0 cells)) can be employed. In general, mammalian cells are employedto effect glycosylation. The N-glycans that are produced in mammaliancells are commonly referred to as complex, high mannose, hybrid-typeN-glycans (see e.g., Drickamer (2006)). These complex N-glycans have astructure that typically has two to six outer branches with asialyllactosamine sequence linked to an inner core structureMan₃GlcNAc₂. A complex N-glycan has at least one branch, and or at leasttwo branches, of alternating GlcNAc and galactose (Gal) residues thatterminate in oligosaccharides such as, for example: NeuNAc-; NeuAc α2,6GalNAc al-; NeuAc α2,3 Gal β1,3 GalNAc al-; and NeuAc α2,3/6 Gal β1,4GlcNAc β1.; In addition, sulfate esters can occur on galactose, GalNAc,and GlcNAc residues. NeuAc can be 0-acetylated or replaced by NeuGl(N-glycolylneuraminic acid). Complex N-glycans may also have intrachainsubstitutions of bisecting GlcNAc and core fucose (Fuc).

Additionally or alternatively, glycosylation can be achieved or modifiedthrough enzymatic means, in vitro. For example, one or moreglycosyltransferases may be employed to add specific saccharide residuesto the native or engineered N-glycan of a binding polypeptide, and oneor more glycosidases may be employed to remove unwanted saccharides fromthe N-linked glycan. Such enzymatic means are well known in the art(see. e.g., WO2007/005786, which is incorporated herein by reference inits entirety).

IV. Immunological Effector Functions and Fc Modifications

In certain embodiments, binding polypeptides may comprise an antibodyconstant region (e.g., an IgG constant region e.g., a human IgG constantregion, e.g., a human IgG1 or IgG4 constant region) which mediates oneor more effector functions. For example, binding of the C1-complex to anantibody constant region may activate the complement system. Activationof the complement system is important in the opsonisation and lysis ofcell pathogens. The activation of the complement system also stimulatesthe inflammatory response and may also be involved in autoimmunehypersensitivity. Further, antibodies bind to receptors on various cellsvia the Fc region (Fc receptor binding sites on the antibody Fc regionbind to Fc receptors (FcRs) on a cell). There are a number of Fcreceptors which are specific for different classes of antibody,including IgG (gamma receptors), IgE (epsilon receptors), IgA (alphareceptors) and IgM (mu receptors). Binding of antibody to Fc receptorson cell surfaces triggers a number of important and diverse biologicalresponses including engulfment and destruction of antibody-coatedparticles, clearance of immune complexes, lysis of antibody-coatedtarget cells by killer cells (called antibody-dependent cell-mediatedcytotoxicity, or ADCC), release of inflammatory mediators, placentaltransfer and control of immunoglobulin production. In some embodiments,the binding polypeptides (e.g., antibodies or antigen binding fragmentsthereof) bind to an Fc-gamma receptor. In alternative embodiments,binding polypeptides may comprise a constant region which is devoid ofone or more effector functions (e.g., ADCC activity) and/or is unable tobind Fcγ receptor.

Certain embodiments include antibodies in which at least one amino acidin one or more of the constant region domains has been deleted orotherwise altered so as to provide desired biochemical characteristicssuch as reduced or enhanced effector functions, the ability tonon-covalently dimerize, increased ability to localize at the site of atumor, reduced serum half-life, or increased serum half-life whencompared with a whole, unaltered antibody of approximately the sameimmunogenicity. For example, certain antibodies for use in thediagnostic and treatment methods described herein are domain deletedantibodies which comprise a polypeptide chain similar to animmunoglobulin heavy chain, but which lack at least a portion of one ormore heavy chain domains. For instance, in certain antibodies, oneentire domain of the constant region of the modified antibody will bedeleted, for example, all or part of the CH2 domain will be deleted.

In certain other embodiments, binding polypeptides comprise constantregions derived from different antibody isotypes (e.g., constant regionsfrom two or more of a human IgG1, IgG2, IgG3, or IgG4). In otherembodiments, binding polypeptides comprises a chimeric hinge (i.e., ahinge comprising hinge portions derived from hinge domains of differentantibody isotypes, e.g., an upper hinge domain from an IgG4 molecule andan IgG1 middle hinge domain). In one embodiment, binding polypeptidescomprise an Fc region or portion thereof from a human IgG4 molecule anda Ser228Pro mutation (EU numbering) in the core hinge region of themolecule.

In certain embodiments, the Fc portion may be mutated to increase ordecrease effector function using techniques known in the art. Forexample, the deletion or inactivation (through point mutations or othermeans) of a constant region domain may reduce Fc receptor binding of thecirculating modified antibody thereby increasing tumor localization. Inother cases it may be that constant region modifications consistent withthe instant invention moderate complement binding and thus reduce theserum half life and nonspecific association of a conjugated cytotoxin.Yet other modifications of the constant region may be used to modifydisulfide linkages or oligosaccharide moieties that allow for enhancedlocalization due to increased antigen specificity or flexibility. Theresulting physiological profile, bioavailability and other biochemicaleffects of the modifications, such as tumor localization,biodistribution and serum half-life, may easily be measured andquantified using well know immunological techniques without undueexperimentation.

In certain embodiments, an Fc domain employed in an antibody is an Fcvariant. As used herein, the term “Fc variant” refers to an Fc domainhaving at least one amino acid substitution relative to the wild-type Fcdomain from which said Fc domain is derived. For example, wherein the Fcdomain is derived from a human IgG1 antibody, the Fc variant of saidhuman IgG1 Fc domain comprises at least one amino acid substitutionrelative to said Fc domain.

The amino acid substitution(s) of an Fc variant may be located at anyposition (i.e., any EU convention amino acid position) within the Fcdomain. In one embodiment, the Fc variant comprises a substitution at anamino acid position located in a hinge domain or portion thereof. Inanother embodiment, the Fc variant comprises a substitution at an aminoacid position located in a CH2 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH3 domain or portion thereof. In anotherembodiment, the Fc variant comprises a substitution at an amino acidposition located in a CH4 domain or portion thereof.

The binding polypeptides may employ any art-recognized Fc variant whichis known to impart an improvement (e.g., reduction or enhancement) ineffector function and/or FcR binding. Said Fc variants may include, forexample, any one of the amino acid substitutions disclosed inInternational PCT Publications WO88/07089A1, WO96/14339A1, WO98/05787A1,WO98/23289A1, WO99/51642A1, WO99/58572A1, WO00/09560A2, WO00/32767A1,WO00/42072A2, WO02/44215A2, WO02/060919A2, WO03/074569A2, WO04/016750A2,WO04/029207A2, WO04/035752A2, WO04/063351A2, WO04/074455A2,WO04/099249A2, WO05/040217A2, WO05/070963A1, WO05/077981A2,WO05/092925A2, WO05/123780A2, WO06/019447A1, WO06/047350A2, andWO06/085967A2 or U.S. Pat. Nos. 5,648,260; 5,739,277; 5,834,250;5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242,195; 6,277,375;6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784,each of which is incorporated in its entirety by reference herein. Inone exemplary embodiment, a binding polypeptide may comprise an Fcvariant comprising an amino acid substitution at EU position 268 (e.g.,H268D or H268E). In another exemplary embodiment, a binding polypeptidemay comprise an amino acid substitution at EU position 239 (e.g., S239Dor S239E) and/or EU position 332 (e.g., I332D or I332Q).

In certain embodiments, a binding polypeptide may comprise an Fc variantcomprising an amino acid substitution which alters theantigen-independent effector functions of the antibody, in particularthe circulating half-life of the binding polypeptide. Such bindingpolypeptides exhibit either increased or decreased binding to FcRn whencompared to binding polypeptides lacking these substitutions, therefore,have an increased or decreased half-life in serum, respectively. Fcvariants with improved affinity for FcRn are anticipated to have longerserum half-lives, and such molecules have useful applications in methodsof treating mammals where long half-life of the administered antibody isdesired, e.g., to treat a chronic disease or disorder. In contrast, Fcvariants with decreased FcRn binding affinity are expected to haveshorter half-lives, and such molecules are also useful, for example, foradministration to a mammal where a shortened circulation time may beadvantageous, e.g. for in vivo diagnostic imaging or in situations wherethe starting antibody has toxic side effects when present in thecirculation for prolonged periods. Fc variants with decreased FcRnbinding affinity are also less likely to cross the placenta and, thus,are also useful in the treatment of diseases or disorders in pregnantwomen. In addition, other applications in which reduced FcRn bindingaffinity may be desired include applications localized to the brain,kidney, and/or liver. In one exemplary embodiment, the altered bindingpolypeptides (e.g., antibodies or antigen binding fragments thereof)exhibit reduced transport across the epithelium of kidney glomeruli fromthe vasculature. In another embodiment, the altered binding polypeptides(e.g., antibodies or antigen binding fragments thereof) exhibit reducedtransport across the blood brain barrier (BBB) from the brain into thevascular space. In one embodiment, an antibody with altered FcRn bindingcomprises an Fc domain having one or more amino acid substitutionswithin the “FcRn binding loop” of an Fc domain. The FcRn binding loop iscomprised of amino acid residues 280-299 (according to EU numbering).Exemplary amino acid substitutions which alter FcRn binding activity aredisclosed in International PCT Publication No. WO05/047327 which isincorporated in its entirety by reference herein. In certain exemplaryembodiments, the binding polypeptides (e.g., antibodies or antigenbinding fragments thereof) comprise an Fc domain having one or more ofthe following substitutions: V284E, H285E, N286D, K290E and 5304D (EUnumbering). In yet other exemplary embodiments, the binding moleculescomprise a human Fc domain with the double mutation H433K/N434F (see,e.g., U.S. Pat. No. 8,163,881).

In other embodiments, binding polypeptides, for use in the diagnosticand treatment methods described herein have a constant region, e.g., anIgG1 or IgG4 heavy chain constant region, which is altered to reduce oreliminate glycosylation. For example, binding polypeptides (e.g.,antibodies or antigen binding fragments thereof) may also comprise an Fcvariant comprising an amino acid substitution which alters theglycosylation of the antibody Fc. For example, said Fc variant may havereduced glycosylation (e.g., N- or O-linked glycosylation). In exemplaryembodiments, the Fc variant comprises reduced glycosylation of theN-linked glycan normally found at amino acid position 297 (EUnumbering). In another embodiment, the antibody has an amino acidsubstitution near or within a glycosylation motif, for example, anN-linked glycosylation motif that contains the amino acid sequence NXTor NXS. In a particular embodiment, the antibody comprises an Fc variantwith an amino acid substitution at amino acid position 228 or 299 (EUnumbering). In more particular embodiments, the antibody comprises anIgG1 or IgG4 constant region comprising an S228P and a T299A mutation(EU numbering).

Exemplary amino acid substitutions which confer reduced or alteredglycosylation are disclosed in International PCT Publication No.WO05/018572, which is incorporated in its entirety by reference herein.In some embodiments, the binding polypeptides are modified to eliminateglycosylation. Such binding polypeptides may be referred to as “agly”binding polypeptides (e.g. “agly” antibodies). While not being bound bytheory, it is believed that “agly” binding polypeptides may have animproved safety and stability profile in vivo. Agly binding polypeptidescan be of any isotype or subclass thereof, e.g., IgG1, IgG2, IgG3, orIgG4. In certain embodiments, agly binding polypeptides comprise anaglycosylated Fc region of an IgG4 antibody which is devoid ofFc-effector function, thereby eliminating the potential for Fc mediatedtoxicity to the normal vital organs that express IL-6. In yet otherembodiments, binding polypeptides comprise an altered glycan. Forexample, the antibody may have a reduced number of fucose residues on anN-glycan at Asn297 of the Fc region, i.e., is afucosylated.Afucosylation increases FcγRII binding on the NK cells and potentlyincreases ADCC. It has been shown that a diabody comprising an anti-IL-6scFv and an anti-CD3 scFv induces killing of IL-6 expressing cells byADCC. Accordingly, in one embodiment, an afucosylated anti-IL-6 antibodyis used to target and kill IL-6-expressing cells. In another embodiment,the binding polypeptide may have an altered number of sialic acidresidues on the N-glycan at Asn297 of the Fc region. Numerousart-recognized methods are available for making “agly” antibodies orantibodies with altered glycans. For example, genetically engineeredhost cells (e.g., modified yeast, e.g., Picchia, or CHO cells) withmodified glycosylation pathways (e.g., glycosyl-transferase deletions)can be used to produce such antibodies.

V. Effector Moieties

In certain embodiments, the binding polypeptides of the currentdisclosure comprise effector moieties (e.g., targeting moieties). Ingeneral these effector moieties are conjugated (either directly orthrough a linker moiety) to an N-linked glycan on the bindingpolypeptide, (e.g., an N-linked glycan linked to N298 (EU numbering) ofthe CH2 domain and/or N114 (Kabat numbering) of a CH1 domain). Incertain embodiments, the binding polypeptide is full length antibodycomprising two CH1 domains with a glycan at Kabat position 114 (EUposition 118), wherein both of the glycans are conjugated to one or moreeffector moieties.

Any effector moiety can be added to the binding polypeptides disclosedherein. The effector moieties can add a non-natural function to analtered antibody or fragments thereof without significantly altering theintrinsic activity of the binding polypeptide. In certain exemplaryembodiments, an effector moiety is a targeting moiety (e.g., aglycopeptide or neoglycan). A modified binding polypeptide (e.g., anantibody) of the current disclosure may comprise one or more effectormoieties, which may be the same of different.

In one embodiment, the effector moiety can be of Formula (I):

H₂N-Q-CON—X  Formula (I),

wherein:

A) Q is NH or O; and

B) CON is a connector moiety; and

C) X is an effector moiety (e.g., a targeting moiety as defined herein).

The connector moiety connects the therapeutic agent to H2N-Q-. Theconnector moiety can include at least one of any suitable componentsknown to those skilled in the art, including, for example, an alkylenylcomponent, a polyethylene glycol component, a poly(glycine) component, apoly(oxazoline) component, a carbonyl component, a component derivedfrom cysteinamide, a component derived from valine coupled withcitruline, and a component derived from 4-aminobenzyl carbamate, or anycombination thereof.

In another embodiment, the effector moiety of Formula (I) can be ofFormula (Ia):

H₂N-Q-CH₂—C(O)—Z—X  Formula (Ia),

wherein:

A) Q is NH or O; and

B) Z is -Cys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f),

-   -   wherein        -   i. Cys is a component derived cysteinamide;        -   ii. MC is a component derived from maleimide;        -   iii. VC is a component derived from valine coupled with            citruline;        -   iv. PABC is a component derived from 4-aminobenzyl            carbamate;        -   v. X is an effector moiety (e.g., a targeting moiety as            defined herein);        -   vi. a is 0 or 1;        -   vii. b is 0 or 1;        -   viii. c is 0 or 1; and        -   ix. f is 0 or 1

The “component derived from cysteinamide” is the point of attachment toH₂N-Q-CH₂—C(O)—. In one embodiment, the “component derived fromcysteinamide” can refer to one or more portions of the effector moietyhaving the structure:

In one embodiment, the “Cys” component of an effector moiety may includeone such portion. For example, the following structure shows an effectormoiety with one such portion (wherein the “Cys” component is indicatedwith the dotted line box):

In another embodiment, the “Cys” component of an effector moiety mayinclude two or more such portions. For example, the following moietycontains two such portions:

As can be seen from the structure, each “Cys” component bears an-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—X group.

In one embodiment, the phrase “component derived from maleimide” canrefer to any portion of the effector moiety having the structure:

wherein d is an integer from 2 to 5. The number of MC componentsincluded in any Cys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—Xgroup in the effector moiety is indicated by subscript “a,” and can be 0or 1 In one embodiment, a is 1. In another embodiment, b is 0.

In one embodiment, the “Cys” component can be connected to the “MC”component via the sulfur atom in the “Cys” component, as indicated withthe dotted line box in the structure below:

In one embodiment, the phrase “component derived from valine coupledwith citruline” can refer to any portion of the effector moiety with thefollowing structure:

The number of VC components included in anyCys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—X group in theeffector moiety is indicated by subscript “b,” and can be 0 or 1. In oneembodiment, b is 1. In another embodiment, b is 0.

In one embodiment, the phrase “component derived from 4-aminobenzylcarbamate” can refer to any portion of the effector moiety with thefollowing structure:

The number of PABC components included in anyCys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—X group in theeffector moiety is indicated by subscript “c,” and can be 0 or 1. In oneembodiment, c is 1. In another embodiment, c is 0.

In one embodiment, “C₁₆H₃₂O₈C₂H₄” refers to the following structure:

The number of C₁₆H₃₂O₈ units included in anyCys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—X group in theeffector moiety is indicated by subscript “f,” In one embodiment, fis 1. In another embodiment, f is 0.

In one embodiment, a is 1, b is 1, c is 1, and f is 0.

a) Therapeutic Effector Moieties

In certain embodiments, the binding polypeptides of the currentdisclosure are conjugated to an effector moiety comprising a therapeuticagent, e.g. a drug moiety (or prodrug thereof) or radiolabeled compound.In one embodiment the therapeutic agent is a cytotoxin. Exemplarycytotoxic therapeutic agents are set forth in Table 1 herein.

TABLE 1 Exemplary cytotoxic therapeutic agents

Further exemplary drug moieties include anti-inflammatory, anti-cancer,anti-infective (e.g., anti-fungal, antibacterial, anti-parasitic,anti-viral, etc.), and anesthetic therapeutic agents. In a furtherembodiment, the drug moiety is an anti-cancer agent. Exemplaryanti-cancer agents include, but are not limited to, cytostatics, enzymeinhibitors, gene regulators, cytotoxic nucleosides, tubulin bindingagents or tubulin inhibitors, proteasome inhibitors, hormones andhormone antagonists, anti-angiogenesis agents, and the like. Exemplarycytostatic anti-cancer agents include alkylating agents such as theanthracycline family of drugs (e.g. adriamycin, carminomycin,cyclosporin-A, chloroquine, methopterin, mithramycin, porfiromycin,streptonigrin, porfiromycin, anthracenediones, and aziridines). Othercytostatic anti-cancer agents include DNA synthesis inhibitors (e.g.,methotrexate and dichloromethotrexate, 3-amino-1,2,4-benzotriazine1,4-dioxide, aminopterin, cytosine β-D-arabinofuranoside,5-fluoro-5′-deoxyuridine, 5-fluorouracil, ganciclovir, hydroxyurea,actinomycin-D, and mitomycin C), DNA-intercalators or cross-linkers(e.g., bleomycin, carboplatin, carmustine, chlorambucil,cyclophosphamide, cis-diammineplatinum(II) dichloride (cisplatin),melphalan, mitoxantrone, and oxaliplatin), and DNA-RNA transcriptionregulators (e.g., actinomycin D, daunorubicin, doxorubicin,homoharringtonine, and idarubicin). Other exemplary cytostatic agentsthat are compatible with the present disclosure include ansamycinbenzoquinones, quinonoid derivatives (e.g. quinolones, genistein,bactacyclin), busulfan, ifosfamide, mechlorethamine, triaziquone,diaziquone, carbazilquinone, indoloquinone EO9,diaziridinyl-benzoquinone methyl DZQ, triethylenephosphoramide, andnitrosourea compounds (e.g. carmustine, lomustine, semustine).

Exemplary cytotoxic nucleoside anti-cancer agents include, but are notlimited to: adenosine arabinoside, cytarabine, cytosine arabinoside,5-fluorouracil, fludarabine, floxuridine, ftorafur, and6-mercaptopurine. Exemplary anti-cancer tubulin binding agents include,but are not limited to: taxoids (e.g. paclitaxel, docetaxel, taxane),nocodazole, rhizoxin, dolastatins (e.g. Dolastatin-10, -11, or -15),colchicine and colchicinoids (e.g. ZD6126), combretastatins (e.g.Combretastatin A-4, AVE-6032), and vinca alkaloids (e.g. vinblastine,vincristine, vindesine, and vinorelbine (navelbine)). Exemplaryanti-cancer hormones and hormone antagonists include, but are notlimited to: corticosteroids (e.g. prednisone), progestins (e.g.hydroxyprogesterone or medroprogesterone), estrogens, (e.g.diethylstilbestrol), antiestrogens (e.g. tamoxifen), androgens (e.g.testosterone), aromatase inhibitors (e.g. aminogluthetimide),17-(allylamino)-17-demethoxygeldanamycin, 4-amino-1,8-naphthalimide,apigenin, brefeldin A, cimetidine, dichloromethylene-diphosphonic acid,leuprolide (leuprorelin), luteinizing hormone-releasing hormone,pifithrin-a, rapamycin, sex hormone-binding globulin, and thapsigargin.Exemplary anti-cancer, anti-angiogenesis compounds include, but are notlimited to: Angiostatin Kl-3, DL-a-difluoromethyl-ornithine, endostatin,fumagillin, genistein, minocycline, staurosporine, and (±)-thalidomide.

Exemplary anti-cancer enzyme inhibitors include, but are not limited to:S(+)-camptothecin, curcumin, (−)-deguelin, 5,6-diCHlorobenz-imidazole1-β-D-ribofuranoside, etoposide, formestane, fostriecin, hispidin,2-imino-1-imidazolidineacetic acid (cyclocreatine), mevinolin,trichostatin A, tyrphostin AG 34, and tyrphostin AG 879.

Exemplary anti-cancer gene regulators include, but are not limited to:5-aza-2′-deoxycytidine, 5-azacytidine, cholecalciferol (vitamin D3),4-hydroxytamoxifen, melatonin, mifepristone, raloxifene, trans-retinal(vitamin A aldehydes), retinoic acid, vitamin A acid, 9-cis-retinoicacid, 13-cis-retinoic acid, retinol (vitamin A), tamoxifen, andtroglitazone.

Other classes of anti-cancer agents include, but are not limited to: thepteridine family of drugs, diynenes, and the podophyllotoxins.Particularly useful members of those classes include, for example,methopterin, podophyllotoxin, or podophyllotoxin derivatives such asetoposide or etoposide phosphate, leurosidine, vindesine, leurosine andthe like.

Still other anti-cancer agents that are compatible with the teachingsherein include auristatins (e.g. auristatin E and monomethylauristan E),geldanamycin, calicheamicin, gramicidin D, maytansanoids (e.g.maytansine), neocarzinostatin, topotecan, taxanes, cytochalasin B,ethidium bromide, emetine, tenoposide, colchicin, dihydroxyanthracindione, mitoxantrone, procaine, tetracaine, lidocaine,propranolol, puromycin, and analogs or homologs thereof.

Still other anti-cancer agents that are compatible with the teachingsherein include tomaymycin derivatives, maytansine derivatives,cryptophycine derivatives, anthracycline derivatives, bisphosphonatederivatives, leptomycin derivatives, streptonigrin derivatives,auristatine derivatives, and duocarmycin derivatives.

Another class of compatible anti-cancer agents that may be used as drugmoieties are radiosensitizing drugs that may be effectively directed totumor or immunoreactive cells. Such drug moeities enhance thesensitivity to ionizing radiation, thereby increasing the efficacy ofradiotherapy. Not to be limited by theory, an antibody modified with aradiosensitizing drug moiety and internalized by the tumor cell woulddeliver the radiosensitizer nearer the nucleus where radiosensitizationwould be maximal. Antibodies which lose the radiosensitizer moiety wouldbe cleared quickly from the blood, localizing the remainingradiosensitization agent in the target tumor and providing minimaluptake in normal tissues. After clearance from the blood, adjunctradiotherapy could be administered by external beam radiation directedspecifically to the tumor, radioactivity directly implanted in thetumor, or systemic radioimmunotherapy with the same modified antibody.

In one embodiment, the therapeutic agent comprises radionuclides orradiolabels with high-energy ionizing radiation that are capable ofcausing multiple strand breaks in nuclear DNA, leading to cell death.Exemplary high-energy radionuclides include: ⁹⁰Y, ¹²⁵I, ¹³¹I, ¹²³I,¹¹¹In, ¹⁰⁵Rh, ¹⁵³Sm, ⁶⁷Cu, ⁶⁷Ga, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re and ¹⁸⁸Re. Theseisotopes typically produce high energy α- or β-particles which have ashort path length. Such radionuclides kill cells to which they are inclose proximity, for example neoplastic cells to which the conjugate hasattached or has entered. They have little or no effect on non-localizedcells and are essentially non-immunogenic. Alternatively, high-energyisotopes may be generated by thermal irradiation of an otherwise stableisotope, for example as in boron neutron-capture therapy (Guan et al.,PNAS, 95: 13206-10, 1998).

In one embodiment, the therapeutic agent is selected from MMAE, MMAF,and PEGS-Dol10.

Exemplary therapeutic effector moieties include the structures:

In one embodiment, the effector moiety is selected from:

In certain embodiments, the effector moiety contains more than onetherapeutic agent. These multiple therapeutic agents can be the same ordifferent.

b) Diagnostic Effector Moieties

In certain embodiments, the binding polypeptides of the currentdisclosure are conjugated to an effector moiety comprising a diagnosticagent. In one embodiment, the diagnostic agent is a detectable smallmolecule label e.g. biotin, fluorophores, chromophores, spin resonanceprobes, or radiolabels. Exemplary fluorophores include fluorescent dyes(e.g. fluorescein, rhodamine, and the like) and other luminescentmolecules (e.g. luminal). A fluorophore may be environmentally-sensitivesuch that its fluorescence changes if it is located close to one or moreresidues in the modified binding polypeptide that undergo structuralchanges upon binding a substrate (e.g. dansyl probes). Exemplaryradiolabels include small molecules containing atoms with one or morelow sensitivity nuclei (¹³C, ¹⁵N, ²H, ¹²⁵I, ¹²⁴I, ¹²³I, ⁹⁹Tc, ⁴³K, ⁵²Fe,⁶⁴Cu, ⁶⁸Ga, ¹¹¹In and the like). The radionuclide can be, e.g., a gamma,photon, or positron-emitting radionuclide with a half-life suitable topermit activity or detection after the elapsed time betweenadministration and localization to the imaging site.

In one embodiment, the diagnostic agent is a polypeptide. Exemplarydiagnostic polypeptides include enzymes with fluorogenic or chromogenicactivity, e.g. the ability to cleave a substrate which forms afluorophore or chromophore as a product (i.e. reporter proteins such asluciferase). Other diagnostic proteins may have intrinsic fluorogenic orchromogenic activity (e.g., green, red, and yellow fluorescentbioluminescent aequorin proteins from bioluminescent marine organisms)or they may comprise a protein containing one or more low-energyradioactive nuclei (¹³C, ¹⁵N, ²H, ¹²⁵I, ¹²⁴I, ¹²³I, ⁹⁹Tc, ⁴³K, ⁵²Fe,⁶⁴Cu, ⁶⁸Ga, ¹¹¹In and the like).

With respect to the use of radiolabeled conjugates in conjunction withthe present disclosure, binding polypeptides of the current disclosuremay be directly labeled (such as through iodination) or may be labeledindirectly through the use of a chelating agent. As used herein, thephrases “indirect labeling” and “indirect labeling approach” both meanthat a chelating agent is covalently attached to a binding polypeptideand at least one radionuclide is associated with the chelating agent.Such chelating agents are typically referred to as bifunctionalchelating agents as they bind both the polypeptide and the radioisotope.Exemplary chelating agents comprise1-isothiocycmatobenzyl-3-methyldiothelene triaminepentaacetic acid(“MX-DTPA”) and cyclohexyl diethylenetriamine pentaacetic acid(“CHX-DTPA”) derivatives. Other chelating agents comprise P-DOTA andEDTA derivatives. Exemplary radionuclides for indirect labeling include111In and 90Y. Most imaging studies utilize 5 mCi 1111n-labeledantibody, because this dose is both safe and has increased imagingefficiency compared with lower doses, with optimal imaging occurring atthree to six days after antibody administration. See, for example,Murray, (1985), J. Nuc. Med. 26: 3328 and Carraguillo et al, (1985), J.Nuc. Med. 26: 67. An exemplary radionuclide for direct labeling is 131I.Those skilled in the art will appreciate that non-radioactive conjugatesmay also be assembled depending on the selected agent to be conjugated.

In certain embodiments, the diagnostic effector moiety is a FRET(Fluorescence Resonance Energy Transfer) probe. FRET has been used for avariety of diagnostic applications including cancer diagnostics. A FRETprobe may include a cleavable linker (enzyme sensitive or pH linker)connecting the donor and acceptor moieties of the FRET probe, whereincleavage results in enhanced fluorescence (including near Infrared)(see, e.g., A. Cobos-Correa et. al. Membrane-bound FRET probe visualizesMMP12 activity in pulmonary inflammation, Nature Chemical Biology(2009), 5(9), 628-63; S. Gehrig et. al. Spatially Resolved Monitoring ofNeutrophil Elastase Activity with Ratiometric Fluorescent Reporters(2012) Angew. Chem. Int. Ed., 51, 6258-6261).

In one embodiment, the effector moiety is selected from:

c) Functionalized Effector Moieties

In certain embodiments, effector moieties may be functionalized tocontain one or more additional groups in addition to the effector moietyitself. For example, the effector moiety may contain cleavable linkerswhich release the effector moiety from the binding polypeptide underparticular conditions. In exemplary embodiments, the effector moiety mayinclude a linker that is cleavable by cellular enzymes and/or is pHsensitive. Additionally or alternatively, the effector moiety maycontain a disulfide bond that cleaved by intracellular glutathione uponuptake into the cell. Exemplary disulfide and pH sensitive linkers areprovided below:

In yet other embodiments, the effector moiety may include hydrophilicand biocompatible moieties such as poly(glycine), poly(oxazoline),and/or PEG moieties. Exemplary structures (“Y”) are provided below:

In certain embodiments, the effector moiety contains an aminooxy groupwhich facilitates conjugation to a binding polypeptide via a stableoxime linkage. Exemplary effector moieties containing aminooxy groupsare set forth in Table 2 herein.

TABLE 2 Exemplary aminoxy effector moieties (wherein X can be anylinker, Y is any spacer, and wherein X and/or Y are optional) Z—Y X Drug

W, W1, and W2 =

Y =

X = Z =

In other embodiments, the effector moiety contains a hydrazide and/orN-alkylated hydrazine group to facilitate conjugation to a bindingpolypeptide via a stable hydrazone linkage. Exemplary effector moietiescontaining aminooxy groups are set forth in Table 14 herein.

TABLE 14 Exemplary hydrazine and/or hydrazide effector moieties

d) Targeting Moieties

In certain embodiments, effector moieties comprise targeting moietiesthat specifically bind to one or more target molecules. Any type oftargeting moiety can be employed including, without limitation,proteins, nucleic acids, lipids, carbohydrates (e.g., glycans), andcombinations thereof (e.g., glycoproteins, glycopeptides, andglycolipids). In certain embodiments, the targeting moiety is acarbohydrate or glycopeptide. In certain embodiments, the targetingmoiety is a glycan. Targeting moieties can be naturally or non-naturallyoccurring molecules. Targeting moieties suitable for conjugation mayinclude those containing aminooxy linkers (see, e.g., FIGS. 40 and 41).

The targeting moieties described in the present invention may bind toany type of cell, including animal (e.g., mammalian), plant, or insectcells either in vitro or in vivo, without limitation. The cells may beof endodermal, mesodermal, or ectodermal origins, and may include anycell type. In certain embodiments, the targeting moiety binds to a cell,e.g., a mammalian cell, a facilitates delivery of a binding polypeptideto the targeted cell, e.g., to improve cell-targeting and/or uptake.Exemplary target cells include, without limitation, immune cells (e.g.,lymphocytes such as B cells, T cells, natural killer (NK) cells,basophils, macrophages, or dendritic cells), liver cells (e.g.,hepatocytes or non-parenchymal cells such as liver sinusoidalendothelial cells, Kupffer cells, or hepatic stellate cells), tumorcells (e.g., any malignant or benign cell including hepatoma cells, lungcancer cells, sarcoma cells, leukemia cells, or lymphoma cells),vascular cells (e.g., aortic endothelial cells or pulmonary arteryendothelial cells), epithelial cells (e.g., simple squamous epithelialcells, simple columnar epithelial cells, pseudostratified columnarepithelial cells, or stratified squamous epithelial cells), ormesenchymal cells (e.g., cells of the lymphatic and circulatory systems,bone, and cartilage cells).

In one embodiment, the binding polypeptide comprising one or moretargeting moieties is internalized by the cell. In another embodiment,the amount of the binding polypeptide comprising one or more targetingmoieties internalized by the cell is greater than the amount of areference binding polypeptide lacking a targeting moiety internalized bythe cell.

In one embodiment, the targeting moiety binds to a receptor on thetarget cell. For example, the targeting moiety may comprise a mannose 6phosphate moiety that binds to a mannose 6 phosphate receptor on thecell. In other exemplary embodiments, the targeting moiety binds to aSiglec on a target cell. Exemplary Siglecs include sialoadhesin(Siglec-1), CD22 (Siglec-2), CD33 (Siglec-3), MAG (Siglec-4), Siglec-5,Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12,Siglec-14, or Siglec-15. In yet other embodiments, the targeting moietycomprises an α2,3-, α2,6-, or α2,8-linked sialic acid residue. In afurther embodiment, the targeting moiety comprises an α2,3-siallylactosemoiety or an α2,6-siallylactose moiety. Other exemplary receptorsinclude lectin receptors, including but not limited to C-type lectinreceptors, galectins, and L-type lectin receptors. Exemplary lectinreceptors include: DEC-205 (CD205; lymphocyte antigen 75), macrophagemannose receptor (MMR; CD206), Dectin-1, Dectin-2, macrophage-inducibleC-type lectin (Mincle), dendritic cell-specific ICAM3-grabbingnonintegrin (DC-SIGN, CD209), DC NK lectin group receptor-1 (DNGR-1),Langerin (CD207), a lectican, an asialoglycoprotein receptor, C-lectinreceptor dendritic cell immunoreceptor (CLEC4A; CLECSF6; DCIR),macrophage galactose-type lectin (MGL), a DC receptor, a collectin, aselectin, an NK-cell receptor, a multi-C-type lectin domain (CTLD)endocytic receptor, a Reg group (type VII) lectin, chondrolectin,tetranectin, polycystin, attractin (ATRN), eosinophil major basicprotein (EMBP), DiGeorge Syndrome Critical Region Gene 2 (DGCR2),Thrombomodulin, Bimlec, a group XVI lectin (SEEC), and a group XVIIlectin (CBCP/Frem1/QBRICK).

The binding polypeptides of the present invention may be used to removetoxic compounds and harmful substances from the liver in multiplediseases by targeting carbohydrate receptors (e.g., mannose 6-phosphatereceptor, mannose receptor, and asialoglycoprotein receptor). Pleasesee: Ganesan, L. P. et al: Rapid and Efficient Clearance of Blood-borneVirus by Liver Sinusoidal Endothelium. PLoS Pathogens 2011, 9: 1; andMonnier, V. M. et al: Glucosepane: a poorly understood advancedglycation end product of growing importance for diabetes and itscomplications. Clin Chem Lab Med 2014; 52: 21.

The binding polypeptides of the present invention may also be used totarget tumor cells through targeting one or more different cellreceptors including, but not limited to: carbohydrate receptors,asialoglycoprotein receptors, and/or Siglecs. Please see: Chen, W. C. etal: In vivo targeting of B-cell lymphoma with glycan ligands of CD22.Blood 2010, 115: 4778; Chen, W. C. et al: Targeting B lymphoma withnanoparticles bearing glycan ligands of CD22. Leuk Lymphoma 2012, 53:208; Hatakeyama, S. et al: Targeted drug delivery to tumor vasculatureby a carbohydrate mimetic peptide. PNAS, 2011, 108: 19587; Hong, F. etal: β-Glucan Functions as an Adjuvant for Monoclonal AntibodyImmunotherapy by Recruiting Tumoricidal Granulocytes as Killer Cells.Cancer Res. 2003, 23: 9023; Kawasakia, N. et al: Targeted delivery oflipid antigen to macrophages via the CD169/sialoadhesin endocyticpathway induces robust invariant natural killer T cell activation. PNAS2013, 110: 7826; and Medina, S. H. et al:N-acetylgalactosamine-functionalized dendrimers as hepatic cancercell-targeted carriers. Biomaterials 2011, 32: 4118.

The binding peptides of the present invention may also be used toregulate immune response through various receptors including, but notlimited to, carbohydrate receptors, DC-SIGNs, and/or Siglecs. Pleasesee: Anthony, R. M. et al: Recapitulation of IVIG Anti-InflammatoryActivity with a Recombinant IgG Fc. Science 2008, 320: 373; Anthony, R.M. et al: Identification of a receptor required for theanti-inflammatory activity of IVIG. PNAS 2008, 105: 19571; Kaneko, Y. etal: Anti-Inflammatory Activity of Immunoglobulin G Resulting from FcSialylation. Science 2006, 313: 670; and Mattner, J. et al: Exogenousand endogenous glycolipid antigens activate NKT cells during microbialinfections. Nature 2005, 434: 525.

In one embodiment, the targeting moiety is a glycopeptide. In a furtherembodiment, the targeting moiety is a tri-galactosylated glycopeptide,e.g., lactose₃-Cys₃Gly₄ (shown in Formula V, below):

In some embodiments, the targeting moiety may be represented by FormulaVII:

In some embodiments, the connector moiety of the effector moiety ofFormula (I) comprises a spacer, including but not limited to a C2-30alkyl or 1 to 32 PEG. In some embodiments, the connector moiety of theeffector moiety is chosen based on approximate length (FIG. 79).Appropriate spacer lengths include but are not limited to ˜12 Å, ˜16 Å,˜20 Å, ˜31 Å, ˜45 Å, ˜60 Å, ˜80 Å, and ˜88 Å.

In certain embodiments, the effector moiety of Formula (I) may berepresented by Formula VIII:

wherein, q is an integer between 1 and 29 inclusive. For example, q maybe 6, 8, 10, 11, 12, 16, 18, or 22. In example embodiments, the effectormoiety of Formula VIII may be represented by:

In other embodiments, the effector moiety of Formula (I) may berepresented by Formula IX:

wherein, p has a value of 1 to 32. For example, p may be 2, 4, 6, 8, 11,12, or 24. In example embodiments, the effector moiety of Formula IX maybe represented by Formula X:

In other embodiments, the effector moiety of Formula (I) may berepresented by Formula XI:

wherein p has a value of 1 to 32. For example, p may be 2, 4, 6, 8, 11,12, or 24. In example embodiments, the effector moiety of Formula XI maybe represented by Formula XII:

e) PEG Moieties

In other aspects, the effector moiety is a moiety comprisingpoly(ethylene glycol) (PEG, PEO, or POE). PEG is an oligomer or polymerof ethylene oxide and has the chemical structure H—(O—CH2-CH2)n-OHwherein the element in parentheses is repeated. PEGylation (orpegylation) is a process in which PEG polymer chains are attached toanother molecule (e.g., a binding polypeptide), which is then describedas PEGylated (or pegylated). PEGylation can serve to reduceimmunogenicity and antigenicity as well as to increase the hydrodynamicsize (size in solution) of the molecule it is attached to, reducingrenal clearance and prolonging circulation time. PEGylation can alsomake molecules more water soluble. In one embodiment of the presentinvention, the PEG moiety may comprise mono-PEG, bi-PEG, or tri-PEG. Inanother embodiment, the PEG moiety comprises 3 to 3.5 PEG.

VI. Conjugation of Effector Moieties to Binding Polypeptides

In certain embodiments, effector moieties are conjugated (eitherdirectly or through a linker moiety) to an oxidized glycan (e.g., anoxidized N-linked glycan) of an altered binding polypeptide, (e.g., anengineered glycan at N114 of an antibody CH1 domain or a native glycanat N297 of an antibody F domain). The term “oxidized glycan” means thatan alcohol substituent on the glycan has been oxidized, providing acarbonyl substituent. The carbonyl substituent can react with suitablenitrogen nucleophile to form a carbon-nitrogen double bond. For example,reaction of the carbonyl group with an aminooxy group or hydrazine groupwould form an oxime or hydrazine, respectively. In one embodiment, thecarbonyl substituent is an aldehyde. Suitable oxidized glycans includeoxidized galactose and oxidized sialic acid.

In one embodiment, the modified polypeptide of Formula (II) may be ofFormula (II):

Ab(Gal-C(O)H)_(x)(Gal-Sia-C(O)H)_(y)  Formula (II),

wherein

A) Ab is an antibody or other binding polypeptide as defined herein;

B) Gal is a component derived from galactose;

C) Sia is a component derived from sialic acid;

D) x is 0 to 5; and

E) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

Any art recognized chemistry can be employed to conjugate an effectormoiety (e.g., an effector moiety comprising a linker moiety) to a glycan(see e.g., Hermanson, G. T., Bioconjugate Techniques. Academic Press(1996), which is incorporated herein ion its entirety). In certainembodiments, a saccharide residue (e.g., a sialic acid or galactoseresidue) of the glycan is first oxidized (e.g., using sodium periodatetreatment of sialic acid or galactose oxidase treatment of galactose) togenerate a reactive aldehyde group. This aldehyde group is reacted witheffector moiety an aminooxy group or hydrazine group to form an oxime orhydrazone linker, respectively. Exemplary methods employing this generalreaction scheme are set forth in Examples 10 to 15.

In certain embodiments, the native or engineered glycans of a bindingpolypeptide are first pre-treated with glycosyltransferase enzymes invitro to provide a terminal saccharide residue that is suitablyreactive. For example, sialylation may be achieved first using acombination of galactosyltransferase (Gal T) and sialyltransferase (SialT). In certain embodiments, biantennary glycans that lack galatose (G0For G0) or that contain only one galactose (G1F or G1) can be convertedto higher-order galactosylated or sialylated structures suitable forconjugation (G1F, G1, G2F, G2, G1S1F, G1S1, G2S1F, G2S1, G2S2F, orG2S2).

An exemplary conjugation scheme for producing sialylated glycoconjugatesis shown in FIG. 25C. In an exemplary embodiment, sialic acid residuesare introduced enzymatically and site specifically into the glycan of anantibody (e.g., a native glycan at Asn-297) using a combination ofgalactosyltransferase (Gal T) and sialyltransferase (Sial T). Introducedsialic acid residues are subsequently oxidized with a low concentrationof sodium periodate to yield reactive sialic acid aldehydes suitablyreactive with linkers (e.g., aminooxy linkers) to generateantibody-effector moiety conjugates (e.g., oxime-linkedantibody-effector moiety conjugates). By controlling the number ofglycan and the number of sialic residues with in vitro remodeling, theskilled artisan has precise control over the drug-antibody ratio (DAR)of the antibody-effector moiety conjugates. For example, if ˜1 sialicacid is added onto a single biantennary glycan (A1F) in each of heavychain, an antibody or binding polypeptide with a DAR of 2 can behomogeneously obtained.

VII. Modified Binding Polypeptides

In certain embodiments, the invention provides modified polypeptideswhich are the product of the conjugating effector moieties areconjugated (either directly or through a linker moiety) to one or moreoxidized glycans (e.g., an oxidized N-linked glycan) of an alteredbinding polypeptide (e.g., an engineered glycan at N114 of an antibodyCH1 domain or a native glycan at N297 of an antibody F domain).

In one embodiment, the binding polypeptide can be of Formula (III):

Ab(Gal-C(H)═N-Q-CON—X)_(x)(Gal-Sia-C(H)═N-Q-CON—X)_(y)  Formula (III),

wherein:

A) Ab is an antibody as defined herein;

B) Q is NH or O;

C) CON is a connector moiety as defined herein;

D) X is a targeting moiety as defined herein;

E) Gal is a component derived from galactose;

F) Sia is a component derived from sialic acid;

G) x is 0 to 5; and

H) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

In one embodiment, the binding polypeptide can be of Formula (III) canbe of Formula (IIIa):

Ab(Gal-C(H)═N-Q-CH₂—C(O)—Z—X)_(x)(Gal-Sia-C(H)═N-Q-CH₂—C(O)—Z—X)_(y)  Formula(IIIa),

wherein:

A) Ab is an antibody;

B) Q is NH or 0;

C) Z is Cys-(MC)_(a)-(VC)_(b)-(PABC)_(c)-(C₁₆H₃₂O₈C₂H₄)_(f)—, wherein

-   -   i. Cys is a component derived cysteinamide;    -   ii. MC is a component derived from maleimide;    -   iii. VC is a component derived from valine coupled with        citruline;    -   iv. PABC is a component derived from 4-aminobenzyl carbamate;    -   v. X is an effector moiety (e.g., a targeting moiety as defined        herein);    -   vi. a is 0 or 1;    -   vii. b is 0 or 1;    -   viii. c is 0 or 1; and    -   ix. f is 0 or 1;

D) X is a therapeutic agent as defined herein;

E) Gal is a component derived from galactose;

F) Sia is a component derived from sialic acid;

G) x is 0 to 5; and

H) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

It is to be understood that the Formula (III) is not intended to implythat the antibody, the Gal substituent, and the Gal-Sia substituent areconnected in a chain-like manner. Rather, when such substituents arepresent, the antibody is connected directly to each substituent. Forexample, a binding polypeptide of Formula (III) in which x is 1 and y is2 could have the arrangement shown below:

The CON substituent in Formula (III) and components therein are asdescribed with regard to Formula (I) for effector moieties.

In one embodiment, Q is NH. In another embodiment, Q is O.

In one embodiment, x is 0.

The antibody Ab of Formula (III) may be any suitable antibody asdescribed herein.

In one embodiment, there is provided a method for preparing the bindingpolypeptide of Formula (III), the method comprising reacting an effectormoiety of Formula (I):

NH₂-Q-CON—X  Formula (I),

wherein:

A) Q is NH or O;

B) CON is a connector moiety; and

C) X is an effector moiety (e.g., a targeting moiety as defined herein),

with a modified antibody of Formula (II)

Ab(OXG)_(r)  Formula (II)

wherein

A) OXG is an oxidized glycan; and

B) r is selected from 0 to 4;

In one embodiment, there is provided a method for preparing the bindingpolypeptide of Formula (III), the method comprising reacting an effectormoiety of Formula (I):

NH₂-Q-CON—X  Formula (I),

wherein:

A) Q is NH or O;

B) CON is a connector moiety; and

C) X is an effector moiety (e.g., a targeting moiety as defined herein),

with a modified antibody of Formula (IIa)

Ab(Gal-C(O)H)_(x)(Gal-Sia-C(O)H)_(y)  Formula (IIIa),

wherein

A) Ab is an antibody as described herein;

B) Gal is a component derived from galactose;

C) Sia is a component derived from sialic acid;

D) x is 0 to 5; and

E) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

VII. Methods of Treatment with Modified Antibodies

In one aspect, the invention provides methods of treating or diagnosinga patient in need thereof comprising administering an effective amountof a binding polypeptide disclosed herein. In certain embodiments of thepresent disclosure provide kits and methods for the diagnosis and/ortreatment of disorders, e.g., neoplastic disorders in a mammaliansubject in need of such treatment. In certain exemplary embodiments, thesubject is a human.

The binding polypeptides of the current disclosure are useful in anumber of different applications. For example, in one embodiment, thesubject binding polypeptides are useful for reducing or eliminatingcells bearing an epitope recognized by the binding domain of the bindingpolypeptide. In another embodiment, the subject binding polypeptides areeffective in reducing the concentration of or eliminating solubleantigen in the circulation. In one embodiment, the binding polypeptidesmay reduce tumor size, inhibit tumor growth and/or prolong the survivaltime of tumor-bearing animals. Accordingly, this disclosure also relatesto a method of treating tumors in a human or other animal byadministering to such human or animal an effective, non-toxic amount ofmodified antibody. One skilled in the art would be able, by routineexperimentation, to determine what an effective, non-toxic amount ofmodified binding polypeptide would be for the purpose of treatingmalignancies. For example, a therapeutically active amount of a modifiedantibody or one or more fragments thereof may vary according to factorssuch as the disease stage (e.g., stage I versus stage IV), age, sex,medical complications (e.g., immunosuppressed conditions or diseases)and weight of the subject, and the ability of the modified antibody toelicit a desired response in the subject. The dosage regimen may beadjusted to provide the optimum therapeutic response. For example,several divided doses may be administered daily, or the dose may beproportionally reduced as indicated by the exigencies of the therapeuticsituation.

In general, the compositions provided in the current disclosure may beused to prophylactically or therapeutically treat any neoplasmcomprising an antigenic marker that allows for the targeting of thecancerous cells by the modified antibody.

VIII. Methods of Administering Modified Antibodies or Fragments Thereof

Methods of preparing and administering binding polypeptides of thecurrent disclosure to a subject are well known to or are readilydetermined by those skilled in the art. The route of administration ofthe binding polypeptides of the current disclosure may be oral,parenteral, by inhalation or topical. The term parenteral as used hereinincludes intravenous, intraarterial, intraperitoneal, intramuscular,subcutaneous, rectal or vaginal administration. While all these forms ofadministration are clearly contemplated as being within the scope of thecurrent disclosure, a form for administration would be a solution forinjection, in particular for intravenous or intraarterial injection ordrip. Usually, a suitable pharmaceutical composition for injection maycomprise a buffer (e.g. acetate, phosphate or citrate buffer), asurfactant (e.g. polysorbate), optionally a stabilizer agent (e.g. humanalbumin), etc. However, in other methods compatible with the teachingsherein, the modified antibodies can be delivered directly to the site ofthe adverse cellular population thereby increasing the exposure of thediseased tissue to the therapeutic agent.

In one embodiment, the binding polypeptide that is administered is abinding polypeptide of Formula (III):

Ab(Gal-C(H)═N-Q-CON—X)_(x)(Gal-Sia-C(H)═N-Q-CON—X)_(y)  Formula (III),

wherein:

A) Ab is an antibody as defined herein;

B) Q is NH or O;

C) CON is a connector moiety as defined herein;

D) X is an effector moiety (e.g., a targeting moiety as defined herein);

E) Gal is a component derived from galactose;

F) Sia is a component derived from sialic acid;

G) x is 0 to 5; and

H) y is 0 to 5,

-   -   wherein at least one of x and y is not 0.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. In the compositions and methods of the current disclosure,pharmaceutically acceptable carriers include, but are not limited to,0.01-0.1 M, e.g., 0.05M phosphate buffer, or 0.8% saline. Other commonparenteral vehicles include sodium phosphate solutions, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers, such as those based on Ringer's dextrose, andthe like. Preservatives and other additives may also be present such asfor example, antimicrobials, antioxidants, chelating agents, and inertgases and the like. More particularly, pharmaceutical compositionssuitable for injectable use include sterile aqueous solutions (wherewater soluble) or dispersions and sterile powders for the extemporaneouspreparation of sterile injectable solutions or dispersions. In suchcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and will typically be preserved against thecontaminating action of microorganisms, such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquidpolyethylene glycol, and the like), and suitable mixtures thereof. Theproper fluidity can be maintained, for example, by the use of a coatingsuch as lecithin, by the maintenance of the required particle size inthe case of dispersion and by the use of surfactants.

Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal and the like. In manycases, isotonic agents will be included, for example, sugars,polyalcohols, such as mannitol, sorbitol, or sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

In any case, sterile injectable solutions can be prepared byincorporating an active compound (e.g., a modified binding polypeptideby itself or in combination with other active agents) in the requiredamount in an appropriate solvent with one or a combination ofingredients enumerated herein, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating theactive compound into a sterile vehicle, which contains a basicdispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, exemplary methods of preparation includevacuum drying and freeze-drying, which yields a powder of an activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof. The preparations for injections areprocessed, filled into containers such as ampoules, bags, bottles,syringes or vials, and sealed under aseptic conditions according tomethods known in the art. Further, the preparations may be packaged andsold in the form of a kit such as those described in co-pending U.S.Ser. No. 09/259,337 and U.S. Ser. No. 09/259,338 each of which isincorporated herein by reference. Such articles of manufacture willtypically have labels or package inserts indicating that the associatedcompositions are useful for treating a subject suffering from, orpredisposed to autoimmune or neoplastic disorders.

Effective doses of the compositions of the present disclosure, for thetreatment of the above described conditions vary depending upon manydifferent factors, including means of administration, target site,physiological state of the patient, whether the patient is human or ananimal, other medications administered, and whether treatment isprophylactic or therapeutic. Usually, the patient is a human butnon-human mammals including transgenic mammals can also be treated.Treatment dosages may be titrated using routine methods known to thoseof skill in the art to optimize safety and efficacy.

For passive immunization with a binding polypeptide, the dosage canrange, e.g., from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5mg/kg (e.g., 0.02 mg/kg, 0.25 mg/kg, 0.5 mg/kg, 0.75 mg/kg, 1 mg/kg, 2mg/kg, etc.), of the host body weight. For example dosages can be 1mg/kg body weight or 10 mg/kg body weight or within the range of 1-10mg/kg, e.g., at least 1 mg/kg. Doses intermediate in the above rangesare also intended to be within the scope of the current disclosure.Subjects can be administered such doses daily, on alternative days,weekly or according to any other schedule determined by empiricalanalysis. An exemplary treatment entails administration in multipledosages over a prolonged period, for example, of at least six months.Additional exemplary treatment regimens entail administration once perevery two weeks or once a month or once every 3 to 6 months. Exemplarydosage schedules include 1-10 mg/kg or 15 mg/kg on consecutive days, 30mg/kg on alternate days or 60 mg/kg weekly. In some methods, two or moremonoclonal antibodies with different binding specificities areadministered simultaneously, in which case the dosage of each antibodyadministered falls within the ranges indicated.

Binding polypeptides of the current disclosure can be administered onmultiple occasions. Intervals between single dosages can be weekly,monthly or yearly. Intervals can also be irregular as indicated bymeasuring blood levels of modified binding polypeptide or antigen in thepatient. In some methods, dosage is adjusted to achieve a plasmamodified binding polypeptide concentration of 1-1000 μg/ml and in somemethods 25-300 μg/ml. Alternatively, binding polypeptides can beadministered as a sustained release formulation, in which case lessfrequent administration is required. For antibodies, dosage andfrequency vary depending on the half-life of the antibody in thepatient. In general, humanized antibodies show the longest half-life,followed by chimeric antibodies and nonhuman antibodies.

The dosage and frequency of administration can vary depending on whetherthe treatment is prophylactic or therapeutic. In prophylacticapplications, compositions containing the present antibodies or acocktail thereof are administered to a patient not already in thedisease state to enhance the patient's resistance. Such an amount isdefined to be a “prophylactic effective dose.” In this use, the preciseamounts again depend upon the patient's state of health and generalimmunity, but generally range from 0.1 to 25 mg per dose, especially 0.5to 2.5 mg per dose. A relatively low dosage is administered atrelatively infrequent intervals over a long period of time. Somepatients continue to receive treatment for the rest of their lives. Intherapeutic applications, a relatively high dosage (e.g., from about 1to 400 mg/kg of antibody per dose, with dosages of from 5 to 25 mg beingmore commonly used for radioimmunoconjugates and higher doses forcytotoxin-drug modified antibodies) at relatively short intervals issometimes required until progression of the disease is reduced orterminated, or until the patient shows partial or complete ameliorationof disease symptoms. Thereafter, the patient can be administered aprophylactic regime.

Binding polypeptides of the current disclosure can optionally beadministered in combination with other agents that are effective intreating the disorder or condition in need of treatment (e.g.,prophylactic or therapeutic). Effective single treatment dosages (i.e.,therapeutically effective amounts) of 90Y-labeled modified antibodies ofthe current disclosure range from between about 5 and about 75 mCi, suchas between about 10 and about 40 mCi. Effective single treatmentnon-marrow ablative dosages of ¹³¹I-modified antibodies range frombetween about 5 and about 70 mCi, or between about 5 and about 40 mCi.Effective single treatment ablative dosages (i.e., may requireautologous bone marrow transplantation) of ¹³¹I-labeled antibodies rangefrom between about 30 and about 600 mCi, such as between about 50 andless than about 500 mCi. In conjunction with a chimeric antibody, owingto the longer circulating half-life vis-a-vis murine antibodies, aneffective single treatment non-marrow ablative dosages of iodine-131labeled chimeric antibodies range from between about 5 and about 40 mCi,such as less than about 30 mCi. Imaging criteria for, e.g., the ¹¹¹Inlabel, are typically less than about 5 mCi.

While the binding polypeptides may be administered as describedimmediately above, it must be emphasized that in other embodimentsbinding may be administered to otherwise healthy patients as a firstline therapy. In such embodiments the binding polypeptides may beadministered to patients having normal or average red marrow reservesand/or to patients that have not, and are not, undergoing. As usedherein, the administration of modified antibodies or fragments thereofin conjunction or combination with an adjunct therapy means thesequential, simultaneous, coextensive, concurrent, concomitant, orcontemporaneous administration or application of the therapy and thedisclosed antibodies. Those skilled in the art will appreciate that theadministration or application of the various components of the combinedtherapeutic regimen may be timed to enhance the overall effectiveness ofthe treatment. For example, chemotherapeutic agents could beadministered in standard, well known courses of treatment followedwithin a few weeks by radioimmunoconjugates of the present disclosure.Conversely, cytotoxin associated binding polypeptides could beadministered intravenously followed by tumor localized external beamradiation. In yet other embodiments, the modified binding polypeptidemay be administered concurrently with one or more selectedchemotherapeutic agents in a single office visit. A skilled artisan(e.g. an experienced oncologist) would be readily be able to discerneffective combined therapeutic regimens without undue experimentationbased on the selected adjunct therapy and the teachings of the instantspecification.

In this regard it will be appreciated that the combination of thebinding polypeptides and the chemotherapeutic agent may be administeredin any order and within any time frame that provides a therapeuticbenefit to the patient. That is, the chemotherapeutic agent and bindingpolypeptides may be administered in any order or concurrently. Inselected embodiments the binding polypeptides of the present disclosurewill be administered to patients that have previously undergonechemotherapy. In yet other embodiments, the binding polypeptides and thechemotherapeutic treatment will be administered substantiallysimultaneously or concurrently. For example, the patient may be giventhe binding polypeptides while undergoing a course of chemotherapy. Insome embodiments the modified antibody will be administered within oneyear of any chemotherapeutic agent or treatment. In other embodimentsthe binding polypeptides will be administered within 10, 8, 6, 4, or 2months of any chemotherapeutic agent or treatment. In still otherembodiments the binding polypeptide will be administered within 4, 3, 2,or 1 week(s) of any chemotherapeutic agent or treatment. In yet otherembodiments the binding polypeptides will be administered within 5, 4,3, 2, or 1 day(s) of the selected chemotherapeutic agent or treatment.It will further be appreciated that the two agents or treatments may beadministered to the patient within a matter of hours or minutes (i.e.substantially simultaneously).

It will further be appreciated that the binding polypeptides of thecurrent disclosure may be used in conjunction or combination with anychemotherapeutic agent or agents (e.g. to provide a combined therapeuticregimen) that eliminates, reduces, inhibits or controls the growth ofneoplastic cells in vivo. Exemplary chemotherapeutic agents that arecompatible with the current disclosure include alkylating agents, vincaalkaloids (e.g., vincristine and vinblastine), procarbazine,methotrexate and prednisone. The four-drug combination MOPP(mechlethamine (nitrogen mustard), vincristine (Oncovin), procarbazineand prednisone) is very effective in treating various types of lymphoma.In MOPP-resistant patients, ABVD (e.g., adriamycin, bleomycin,vinblastine and dacarbazine), ChIVPP (CHlorambucil, vinblastine,procarbazine and prednisone), CABS (lomustine, doxorubicin, bleomycinand streptozotocin), MOPP plus ABVD, MOPP plus ABV (doxorubicin,bleomycin and vinblastine) or BCVPP (carmustine, cyclophosphamide,vinblastine, procarbazine and prednisone) combinations can be used.Arnold S. Freedman and Lee M. Nadler, Malignant Lymphomas, in HARRISON'SPRINCIPLES OF INTERNAL MEDICINE 1774-1788 (Kurt J. Isselbacher et al,eds., 13th ed. 1994) and V. T. DeVita et al, (1997) and the referencescited therein for standard dosing and scheduling. These therapies can beused unchanged, or altered as needed for a particular patient, incombination with one or more binding polypeptides of the currentdisclosure as described herein.

Additional regimens that are useful in the context of the presentdisclosure include use of single alkylating agents such ascyclophosphamide or chlorambucil, or combinations such as CVP(cyclophosphamide, vincristine and prednisone), CHOP (CVP anddoxorubicin), C-MOPP (cyclophosphamide, vincristine, prednisone andprocarbazine), CAP-BOP (CHOP plus procarbazine and bleomycin), m-BACOD(CHOP plus methotrexate, bleomycin and leucovorin), ProMACE-MOPP(prednisone, methotrexate, doxorubicin, cyclophosphamide, etoposide andleucovorin plus standard MOPP), ProMACE-CytaBOM (prednisone,doxorubicin, cyclophosphamide, etoposide, cytarabine, bleomycin,vincristine, methotrexate and leucovorin) and MACOP-B (methotrexate,doxorubicin, cyclophosphamide, vincristine, fixed dose prednisone,bleomycin and leucovorin). Those skilled in the art will readily be ableto determine standard dosages and scheduling for each of these regimens.CHOP has also been combined with bleomycin, methotrexate, procarbazine,nitrogen mustard, cytosine arabinoside and etoposide. Other compatiblechemotherapeutic agents include, but are not limited to,2-Chlorodeoxyadenosine (2-CDA), 2′-deoxycoformycin and fludarabine.

For patients with intermediate- and high-grade NHL (non-Hodgkin'slymphoma), who fail to achieve remission or relapse, salvage therapy isused. Salvage therapies employ drugs such as cytosine arabinoside,carboplatin, cisplatin, etoposide and ifosfamide given alone or incombination. In relapsed or aggressive forms of certain neoplasticdisorders the following protocols are often used: IMVP-16 (ifosfamide,methotrexate and etoposide), MIME (methyl-gag, ifosfamide, methotrexateand etoposide), DHAP (dexamethasone, high dose cytarabine andcisplatin), ESHAP (etoposide, methylpredisolone, HD cytarabine,cisplatin), CEPP(B) (cyclophosphamide, etoposide, procarbazine,prednisone and bleomycin) and CAMP (lomustine, mitoxantrone, cytarabineand prednisone) each with well-known dosing rates and schedules.

The amount of chemotherapeutic agent to be used in combination with themodified antibodies of the current disclosure may vary by subject or maybe administered according to what is known in the art. See for example,Bruce A Chabner et al, Antineoplastic Agents, in GOODMAN & GILMAN'S THEPHARMACOLOGICAL BASIS OF THERAPEUTICS 1233-1287 ((Joel G. Hardman et al,eds., 9th ed. 1996).

As previously discussed, the binding polypeptides of the presentdisclosure, immunoreactive fragments or recombinants thereof may beadministered in a pharmaceutically effective amount for the in vivotreatment of mammalian disorders. In this regard, it will be appreciatedthat the disclosed binding polypeptides will be formulated to facilitateadministration and promote stability of the active agent.

A pharmaceutical compositions in accordance with the present disclosurecan comprise a pharmaceutically acceptable, non-toxic, sterile carriersuch as physiological saline, nontoxic buffers, preservatives and thelike. For the purposes of the instant application, a pharmaceuticallyeffective amount of the modified binding polypeptide, immunoreactivefragment or recombinant thereof, conjugated or unconjugated to atherapeutic agent, shall be held to mean an amount sufficient to achieveeffective binding to an antigen and to achieve a benefit, e.g., toameliorate symptoms of a disease or disorder or to detect a substance ora cell. In the case of tumor cells, the modified binding polypeptide caninteract with selected immunoreactive antigens on neoplastic orimmunoreactive cells and provide for an increase in the death of thosecells. Of course, the pharmaceutical compositions of the presentdisclosure may be administered in single or multiple doses to providefor a pharmaceutically effective amount of the modified bindingpolypeptide.

In keeping with the scope of the present disclosure, the bindingpolypeptides of the disclosure may be administered to a human or otheranimal in accordance with the aforementioned methods of treatment in anamount sufficient to produce a therapeutic or prophylactic effect. Thebinding polypeptides of the disclosure can be administered to such humanor other animal in a conventional dosage form prepared by combining theantibody of the disclosure with a conventional pharmaceuticallyacceptable carrier or diluent according to known techniques. It will berecognized by one of skill in the art that the form and character of thepharmaceutically acceptable carrier or diluent is dictated by the amountof active ingredient with which it is to be combined, the route ofadministration and other well-known variables. Those skilled in the artwill further appreciate that a cocktail comprising one or more speciesof binding polypeptides described in the current disclosure may prove tobe particularly effective.

IX. Expression of Binding Polypeptides

In one aspect, the invention provides polynucleotides encoding thebinding polypeptides disclosed herein. A method of making a bindingpolypeptide comprising expressing these polynucleotides are alsoprovided.

Polynucleotides encoding the binding polypeptides disclosed herein aretypically inserted in an expression vector for introduction into hostcells that may be used to produce the desired quantity of the claimedantibodies, or fragments thereof. Accordingly, in certain aspects, theinvention provides expression vectors comprising polynucleotidesdisclosed herein and host cells comprising these vectors andpolynucleotides.

The term “vector” or “expression vector” is used herein for the purposesof the specification and claims, to mean vectors used in accordance withthe present invention as a vehicle for introducing into and expressing adesired gene in a cell. As known to those skilled in the art, suchvectors may easily be selected from the group consisting of plasmids,phages, viruses and retroviruses. In general, vectors compatible withthe instant invention will comprise a selection marker, appropriaterestriction sites to facilitate cloning of the desired gene and theability to enter and/or replicate in eukaryotic or prokaryotic cells.

Numerous expression vector systems may be employed for the purposes ofthis invention. For example, one class of vector utilizes DNA elementswhich are derived from animal viruses such as bovine papilloma virus,polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses(RSV, MMTV or MOMLV), or SV40 virus. Others involve the use ofpolycistronic systems with internal ribosome binding sites.Additionally, cells which have integrated the DNA into their chromosomesmay be selected by introducing one or more markers which allow selectionof transfected host cells. The marker may provide for prototrophy to anauxotrophic host, biocide resistance (e.g., antibiotics) or resistanceto heavy metals such as copper. The selectable marker gene can either bedirectly linked to the DNA sequences to be expressed, or introduced intothe same cell by cotransformation. Additional elements may also beneeded for optimal synthesis of mRNA. These elements may include signalsequences, splice signals, as well as transcriptional promoters,enhancers, and termination signals. In some embodiments the clonedvariable region genes are inserted into an expression vector along withthe heavy and light chain constant region genes (such as human genes)synthesized as discussed above.

In other embodiments the binding polypeptides featured in the inventionmay be expressed using polycistronic constructs. In such expressionsystems, multiple gene products of interest such as heavy and lightchains of antibodies may be produced from a single polycistronicconstruct. These systems advantageously use an internal ribosome entrysite (IRES) to provide relatively high levels of polypeptides ineukaryotic host cells. Compatible IRES sequences are disclosed in U.S.Pat. No. 6,193,980 which is incorporated by reference herein. Thoseskilled in the art will appreciate that such expression systems may beused to effectively produce the full range of polypeptides disclosed inthe instant application.

More generally, once a vector or DNA sequence encoding an antibody, orfragment thereof, has been prepared, the expression vector may beintroduced into an appropriate host cell. That is, the host cells may betransformed. Introduction of the plasmid into the host cell can beaccomplished by various techniques well known to those of skill in theart. These include, but are not limited to, transfection (includingelectrophoresis and electroporation), protoplast fusion, calciumphosphate precipitation, cell fusion with enveloped DNA, microinjection,and infection with intact virus. See, Ridgway, A. A. G. “MammalianExpression Vectors” Chapter 24.2, pp. 470-472 Vectors, Rodriguez andDenhardt, Eds. (Butterworths, Boston, Mass. 1988). The transformed cellsare grown under conditions appropriate to the production of the lightchains and heavy chains, and assayed for heavy and/or light chainprotein synthesis. Exemplary assay techniques include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), orfluorescence-activated cell sorter analysis (FACS), immunohistochemistryand the like.

As used herein, the term “transformation” shall be used in a broad senseto refer to the introduction of DNA into a recipient host cell thatchanges the genotype and consequently results in a change in therecipient cell.

Along those same lines, “host cells” refers to cells that have beentransformed with vectors constructed using recombinant DNA techniquesand encoding at least one heterologous gene. In descriptions ofprocesses for isolation of polypeptides from recombinant hosts, theterms “cell” and “cell culture” are used interchangeably to denote thesource of antibody unless it is clearly specified otherwise. In otherwords, recovery of polypeptide from the “cells” may mean either fromspun down whole cells, or from the cell culture containing both themedium and the suspended cells.

In one embodiment, the host cell line used for antibody expression is ofmammalian origin; those skilled in the art can determine particular hostcell lines which are best suited for the desired gene product to beexpressed therein. Exemplary host cell lines include, but are notlimited to, DG44 and DUXB11 (Chinese Hamster Ovary lines, DHFR minus),HELA (human cervical carcinoma), CVI (monkey kidney line), COS (aderivative of CVI with SV40 T antigen), R1610 (Chinese hamsterfibroblast) BALBC/3T3 (mouse fibroblast), HAK (hamster kidney line),SP2/O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI(human lymphocyte), 293 (human kidney). In one embodiment, the cell lineprovides for altered glycosylation, e.g., afucosylation, of theantibodyexpressed therefrom (e.g., PER.C6® (Crucell) or FUT8-knock-outCHO cell lines (Potelligent® Cells) (Biowa, Princeton, N.J.)). In oneembodiment NS0 cells may be used. Host cell lines are typicallyavailable from commercial services, the American Tissue CultureCollection or from published literature.

In vitro production allows scale-up to give large amounts of the desiredpolypeptides. Techniques for mammalian cell cultivation under tissueculture conditions are known in the art and include homogeneoussuspension culture, e.g. in an airlift reactor or in a continuousstirrer reactor, or immobilized or entrapped cell culture, e.g. inhollow fibers, microcapsules, on agarose microbeads or ceramiccartridges. If necessary and/or desired, the solutions of polypeptidescan be purified by the customary chromatography methods, for example gelfiltration, ion-exchange chromatography, chromatography overDEAE-cellulose and/or (immuno-) affinity chromatography.

One or more genes encoding binding polypeptides can also be expressednon-mammalian cells such as bacteria or yeast or plant cells. In thisregard it will be appreciated that various unicellular non-mammalianmicroorganisms such as bacteria can also be transformed; i.e. thosecapable of being grown in cultures or fermentation. Bacteria, which aresusceptible to transformation, include members of theenterobacteriaceae, such as strains of Escherichia coli or Salmonella;Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, andHaemophilus influenzae. It will further be appreciated that, whenexpressed in bacteria, the polypeptides can become part of inclusionbodies. The polypeptides must be isolated, purified and then assembledinto functional molecules.

In addition to prokaryotes, eukaryotic microbes may also be used.Saccharomyces cerevisiae, or common baker's yeast, is the most commonlyused among eukaryotic microorganisms although a number of other strainsare commonly available. For expression in Saccharomyces, the plasmidYRp7, for example, (Stinchcomb et al., Nature, 282:39 (1979); Kingsmanet al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) iscommonly used. This plasmid already contains the TRP1 gene whichprovides a selection marker for a mutant strain of yeast lacking theability to grow in tryptophan, for example ATCC No. 44076 or PEP4-1(Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as acharacteristic of the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan.

Examples

The present invention is further illustrated by the following exampleswhich should not be construed as further limiting. The contents of theSequence Listing, figures and all references, patents, and publishedpatent applications cited throughout this application are expresslyincorporated herein by reference.

Example 1. Design, Preparation, and Characterization of 2C3 Anti-CD-52Hyperglycosylation Antibody Mutants

Multiple hyperglycosylation mutations were designed in the heavy chainof the anti-CD-52 antibody, 2C3, for the purpose of adding a bulky groupto an interaction interface (e.g., the FcRn binding site to modulateantibody pharmacokinetics), for modulating antibody effector function bychanging its interaction with FcγRs, or to introduce a novelcross-linking site subsequence chemical modification for effector moietyconjugation, including but not limited to, drugs, toxins, cytotoxicagents, radionucleotides and the like. The hyperglycosylated 2C3 mutantsare set forth in Table 3.

TABLE 3 Hyperglycosylated 2C3 anti-CD-52 mutants Mutation DesiredBenefit Applications A114N Glycosylation at Asn-Ser-Thr 1) Control 2)Effector moiety conjugation Y436T Glycosylation at Asn434 1) Transplantand other indications Inhibition of FcRn binding which need shorthalf-life Y436S Glycosylation at Asn434 1) Transplant and otherindications Inhibition of FcRn binding which need short half-life S440NGlycosylation at Asn-Leu-Ser 1) Control 2) Effector moiety conjugationS442N Glycosylation at Asn-Leu-Ser 1) Control 2) Effector moietyconjugation Add NGT to Glycosylation 1) Control C-terminal 2) Effectormoiety conjugation S298N/Y300S Glycosylation at Asn298 1) Reduceeffector function Reduced effector function 2) Effector moietyconjugation

1A. Creation of 2C3 Anti-CD-52 Antibody Hyperglycosylation Mutants

The A114N mutation, designated based upon the Kabat numbering system(equivalent to EU position 118), was introduced into the CH1 domain of2C3 by mutagenic PCR. To create the full-length antibody, the VH domainplus the mutated A114N residue was inserted by ligation independentcloning (LIC) into the pENTR-LIC-IgG1 vector encoding antibody CHdomains 1-3. All other mutations were introduced on pENTR-LIC-IgG1 bysite-directed mutagenesis with a QuikChange site-directed mutagenesiskit (Agilent Technologies, Inc., Santa Clara, Calif., USA). The WT 2C3VH was cloned into mutated vectors by LIC. Full-length mutants werecloned into the pCEP4(-E+I)Dest expression vector by Gateway cloning. Fcmutations were designated based on the EU numbering system. Mutationswere confirmed by DNA sequencing. Amino acid sequences of the WT 2C3heavy and light chains and the mutated 2C3 heavy chains are set forth inTable 4. Mutated amino acids are shaded and the consensus glycosylationtarget sites created by the mutation are underlined.

TABLE 4 Amino acid sequences of 2C3 anti-CD-52 antibodies SEQ ID NO NameAmino Acid Sequence 1 Anti-CD-52 DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYWT LNWLLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSG light chainTDFTLKISRVEAEDVGVYYCVQGTHLHTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 2 Anti-CD-52 VQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN WTWVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR heavy chainFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 3 Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNA114N WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR heavy chainFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW GQGTTVTVSSNSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 4 Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNY436S heavy WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR chainFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHSTQKSLSLSPGK 5 Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNS440N heavy WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR chainFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKNLSLSPGK 6 Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNS442N heavy WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR chainFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLNLSPGK 7 Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNNGT WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR heavy chainFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW GQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKNGT 8 Anti-CD-52EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMN S298N/WVRQAPGKGLEWVGQIRLKSNNYATHYAESVKGR Y300SFTISRDDSKNSLYLQMNSLKTEDTAVYYCTPVDFW heavy chainGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG CLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTL MISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYN NTSRVVSVLTVLHQDWLNGKEY KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

The mutants and WT control were transfected into HEK293-EBNA cells in a6-well plate format. As shown in FIG. 9A, the expression level was foundto be ˜0.1 μg/ml, as analyzed by SDS-PAGE and Western blot. Expressionof mutants in conditioned media was also measured by protein A captureon Biacore. Concentration was determined using the dissociation response6 minutes after injection into immobilized Protein A. CHO-produced WT2C3 serially diluted in media from 90 μg/mL to 1.5 ng/mL was used as astandard curve. Concentrations were calculated down to ˜0.2 μg/mL by acalibration curve using a 4-parameter fit. As shown in FIG. 9B, relativeexpressions levels were low and generally corresponded with the Westernblot results.

1B. Verification of Hyperglycosylation

To determine whether additional glycosylation sites were introduced bymutation, 2C3 mutant and wild-type proteins were treated with theuniversal deglycosylating enzyme PNGase F and protein samples wereanalyzed by SDS-PAGE and Western blot. As shown in FIG. 10, only theA114N mutant had an increased apparent molecular weight, indicating thepresence of an additional N-linked carbohydrate.

Small scale antibody preparations were produced to purify the 2C3mutants for further verification of glycosylation site introduction. Asshown in FIG. 11, it was confirmed by SDS-PAGE that only the A114Nmutant had additional glycosylation sites introduced.

1C. Binding Properties of 2C3 Anti-CD-52 Mutants

Biacore was used to compare the binding properties of the purifiedproteins. Mouse and SEC-purified human FcRn-HPC4 were immobilized on aCMS chip via amine coupling. Each antibody was diluted to 200, 50, and10 nM and injected over the immobilized Fc receptors. Campath,CHO-produced WT 2C3, and DEPC-treated Campath were included as positiveand negative controls. As shown in FIG. 13, the Y436S mutant displayedabout a 2-fold decrease in binding to human FcRn. Interestingly, bindingof this mutant to mouse FcRn was not affected. None of the other 2C3mutations had any considerable effect on human or mouse FcRn binding.

Biacore was used to compare the antigen binding properties of thepurified proteins using the CD-52 peptide 741 Biacore binding assay.CD-52 peptide 741 and control peptide 777 were immobilized to a CMSchip. Antibodies were serially diluted 2-fold from 60 to 0.2 nM inHBS-EP and injected in duplicate for 3 min followed by a 5 mindissociation in buffer at a 50 μL/min flow-rate. GLD52 lot 17200-084 wasincluded as a control. The surface was regenerated with 1 pulse of 40 mMHCl. A 1:1 binding model was used to fit the 7.5 to 0.2 nM curves. Asshown in FIG. 16, the A114N mutant had a slightly lower CD-52 bindingaffinity while the NGT mutant had a slightly higher affinity than therest of the mutants in this assay. The CD-52 peptide 741 Biacore bindingassay was repeated with protein purified from larger scale prep. Asshown in FIG. 17, the A114N mutant exhibited CD-52 peptide binding thatwas comparable to WT 2C3.

1D. Charge Characterization of the A114N Mutant

Isoelectric focusing (IEF) was performed to characterize the charge ofthe 2C3 mutants. Purified protein was run on immobilized pH gradient(pH3-10) acrylamide (IPG) gels. As shown in FIG. 18A, A114N was found tohave more negative charges, likely due to sialic acid residues. IntactMS data confirmed the complex structure with sialic acids on the A114Nmutant. In contrast, the WT 2C3 was shown to have G0F and G1F as thedominant glycosylation species (FIGS. 18C and 18D, respectively).

Example 2. Preparation of Hyperglycosylation Mutants in Several AntibodyBackbones

In addition to the 2C3 anti-CD-52 antibody, the A114N mutation wasengineered in several other antibody backbones to confirm that theunique hyperglycosylation site could be introduced into unrelated heavychain variable domain sequences. The hyperglycosylated anti-TEM1,anti-FAP, and anti-Her2 mutants are set forth in Table 5.

TABLE 5 A114N and/or S298N mutants designed in several unrelatedantibody backbones Mutation Antibody Desired benefits Applications A114Nanti-TEM1 Additional glycosylation site at 1) Control anti-FAP the elbowhinge of heavy chain 2) Aminooxy toxin anti-Her2 for site-specificcarbohydrate- conjugation via exposed mediated conjugation sialic acidor galactose group (SAM or GAM) S298N/T299A/Y300S anti-Her2 Switch theglycosylation from 1) Aminooxy toxin (NNAS) Asn297 to an engineeredconjugation via exposed Asn298. Expect solvent sialic acid or galactoseexposed and complex group (SAM or GAM) carbohydrates at S298N, 2)Reduced effector offering conjugation site and function means to removeeffector function A114N/NNAS anti-Her2 Potential for increased 1)Control conjugation yield with two 2) Aminooxy toxin conjugation sitesconjugation via exposed sialic acid or galactose group (SAM or GAM)

2A. Creation of Anti-TEM1 and Anti-FAP Antibody HyperglycosylationMutants

The A114N mutation, designated based upon the Kabat numbering system,was introduced into the CH1 domain of anti-TEM1 and anti-FAP bymutagenic PCR. To create the full-length antibody, the mutated VH plusresidue 114 was inserted by ligation independent cloning (LIC) into thepENTR-LIC-IgG1 vector encoding antibody CH domains 1-3. Full-lengthmutants were then cloned into the pCEP4(-E+I)Dest expression vector byGateway cloning. Mutations were confirmed by DNA sequencing. Amino acidsequences of the anti-TEM1 wild-type and mutated heavy and light chainsare set forth in Table 6. Mutated amino acids are shaded and theconsensus glycosylation target sites created by the mutation areunderlined.

TABLE 6 Amino acid sequences of anti-TEM1 and anti-FAP antibodiesSEQ ID NO Name Amino Acid Sequence 9 Anti-TEM1EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWY WT lightQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTL chainTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRT (clone #187)VAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 10 Anti-TEM1QVQLQESAPGLVKPSETLSLTCTVSGGSIRSYYWSW WT heavyIRQPPGKGLEYIGYIYYTGSAIYNPSLQSRVTISVDTS chainKNQFSLKLNSVTAADTAVYYCAREGVRGASGYYY (clone #187)YGMDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 11 Anti-TEM1QVQLQESAPGLVKPSETLSLTCTVSGGSIRSYYWSW A114NIRQPPGKGLEYIGYIYYTGSAIYNPSLQSRVTISVDTSKNQFSLKLNSVTAADTAVYYCAREGVRGASGYYY YGMDVWGQGTTVTVSSNSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFP AVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQD WLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK*

The mutants and wild-type control were transfected into HEK293-EBNAcells in a triple flask format and purified on HiTrap protein A columns(GE Healthcare Biosciences, Pittsburgh, Pa., USA). As analyzed by A280on a NanoDrop spectrophotometer, the expression of anti-FAP A114N andanti-FAP A114C was about 3 μg/ml and about 1 μg/ml, respectively. Theexpression of anti-TEM1 A114N was about 0.04n/ml.

2B. Verification of Hyperglycosylation

To confirm that the additional glycosylation site was introduced intothe A114N mutants, purified protein from the A114N mutants was analyzedon reducing SDS-PAGE along with wild-type control protein. Oneadditional glycosylation site would add 2000-3000 Daltons to themolecular weight of the heavy chain. As shown in FIG. 20, SDS-PAGEindicated that the anti-FAP and anti-TEM1 A114N mutants heavy chainbands had increased apparent molecular weight, consistent withsuccessful introduction of an additional glycosylation site to bothantibodies.

2C. Creation of Anti-Her2 Antibody Hyperglycosylation Mutants

The Her-2 A114N, Her-2 A114N/NNAS, and WT Her-2 antibodies were createdby ligation independent cloning. The VH domain of Herceptin wassynthesized and PCR-amplified with two LIC-compatible sets of primers,either WT or bearing the A114N mutation. To obtain a full-lengthantibody, amplified VH inserts (WT or A114N) were cloned into two pENTRvectors encoding CH 1-3 domains, pENTR-LIC-IgG1 WT and pENTR-LIC-IgG1NNAS, resulting in three full-length mutants (A114N, NNAS, A114N/NNAS)and WT control as entry clones on pENTR. These mutants were cloned intothe pCEP4(-E+I)Dest expression vector, by Gateway cloning. Mutationswere confirmed by DNA sequencing. Amino acid sequences of the anti-Her-2wild-type and mutated heavy and light chains are set forth in Table 7.Mutated amino acids are shaded and the consensus glycosylation targetsites created by the mutation are underlined.

TABLE 7 Amino acid sequences of anti-Her-2 antibodies SEQ ID NO NameAmino Acid Sequence 12 Anti-Her-2 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAW WTYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFT light chainLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK VQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC 13 Anti-Her-2EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW WTVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS heavy chainADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY AMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 14 Anti-Her-2EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW A114NVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS heavy chainADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY AMDYWGQGTLVTVSSNSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 15 Anti-Her2EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW NNASVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS heavy chainADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY AMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK 16 Anti-Her2EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHW A114N/VRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTIS NNASADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFY heavy chainAMDYWGQGTLVTVSSNSTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWL NGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQ PENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

2D. Expression of the A114N Anti-Her2 Antibody Hyperglycosylation Mutant

The A114N anti-Her2 and wild-type constructs were transfected withLipofectamine-2000 (2.5:1 ratio of reagent to DNA) and XtremeGene HP(3:1 ratio of reagent to DNA) into HEK293-EBNA cells in 12 tripleflasks. Octet measurement of aliquots from day 3 conditioned media (CM)showed that protein expression was consistent across 6 flasks for bothLipofectamine-2000 and XtremeGene HP. As shown in Table 8, the overalltransfection efficiency was about 30% higher with XtremeGene HP.Conditioned media collected on day 3 was pooled together for bothtransfection conditions and purified by protein A column. Octetmeasurement showed 1.8 ug/ml antibody in the serum-containing mock mediaversus 0 ug/ml in no serum mock media.

TABLE 8 A114N anti-Her2 hyperglycosylation mutant expressionLipofectamine- XtremeGene 2000 HP Purified protein Concentration 1.723.18 from protein A (mg/ml) column Volume (ml) 3.5 3.5 Total protein6.02 11.13 (mg) Buffer-exchanged Concentration 15.59 16.86 protein(mg/ml) Volume (ml) 0.2 0.36 Total protein 3.1 6.07 (mg) % Recovery 51.854.5

Conditioned media from day 6 was collected and purified separately foreach transfection condition. Both eluates were buffer-exchangedseparately into PBS, pH 7.2, and concentrated ˜15-fold using Amicon-4(50 kD cut-off) columns. Day 6 CM showed higher expression levelcompared to Day 3 CM. As shown in Table 8, a total of 3 mg of HerceptinA114N 15.59 mg/ml (from Lipofectamine transfection) and 6 mg ofHerceptin A114N 16.86 mg/ml (from XtremeGene HP transfection) wasproduced from day 6 conditioned media for additional downstreamapplications, such as antibody-drug conjugation.

2E. SDS-PAGE and HIC Analysis of the A114N Anti-Her2 Mutant

Prior to conjugation, purified A114N Herceptin was characterized bySDS-PAGE and HIC (hydrophobic interaction chromatography). As shown inFIG. 21, the quality of purified A114N Herceptin was determined to besuitable for further downstream applications.

2F. Conjugation to Engineered Glycosylation

It was demonstrated that: a) a glycosylation site was introduced atKabat position 114 (EU position 118) site on anti-TEM1; b) the A114Nmutant had hyperglycosylation on the heavy chain by reducing SDS-PAGE;and c) the A114N hyperglycosylated mutant had complex carbohydratestructure by intact LC/MS, including terminal sialic acids andgalactose, which are ideal for SAM and GAM conjugation. To confirm thatthe engineered glycosylation site was suitable for conjugation,anti-TEM1 A114N was conjugated with a 5 kDa PEG via aminooxy chemistry.As shown in FIG. 22, PEG was successfully conjugated to anti-TEM1 A114Nthrough an aminooxy linkage. This mutant was also successfully preparedon the anti-FAP and anti-CD-52 2C3 backbones (not shown). These datademonstrate that the glycosylation site at N114 is useful forconjugation of effector moieties.

Example 3: Generation of S298N/Y300S Fe Mutants

Engineered Fc variants were designed and generated in which a newglycosylation site was introduced at EU position Ser 298, next to thenaturally-occurring Asn297 site. The glycosylation at Asn297 was eithermaintained or ablated by mutation. Mutations and desired glycosylationresults are set forth in Table 9.

TABLE 9 Glycosylation states of various antibody variants # MutationDesired Glycosylation State Applications 17 N297Q No glycosylation(agly) Agly Control 18 T299A No glycosylation (agly) Agly Control,unknown effector function 19 N297Q/S298N/Y300S No glycosylation at 297but Reduced effector (NSY) engineered glycosylation site at function;Conjugation 298 via exposed sialic acid or galactose groups. 20S298N/T299A/Y300S No glycosylation at 297 but Reduced effector (STY)engineered glycosylation site at function; Conjugation 298 via exposedsialic acid or galactose groups. 21 S298N/Y300S (SY) Two potentialglycosylation Reduced effector sites at 297 & 298; Alterations function;Conjugation in glycosylation pattern. via exposed sialic acid orgalactose groups. 22 Wild-type 297 control

3A. Creation of H66 αβ-TCR Antibody Altered Glycosylation Variants

Mutations were made on the heavy chain of αβ T-cell receptor antibodyclone #66 by Quikchange using a pENTR_LIC_IgG1 template. The VH domainof HEBE1 Δab IgG1 #66 was amplified with LIC primers before being clonedinto mutated or wild-type pENTR_LIC_IgG1 by LIC to create full-lengthmutant or wild-type antibodies. The subcloning was verified withDraIII/XhoI double digest, producing an approximately 1250 bp-sizedinsert in the successful clones. Those full-length mutants were thencloned into an expression vector, pCEP4(-E+I)Dest, via Gateway cloning.The mutations were confirmed by DNA sequencing. Amino acid sequences ofthe WT H66 anti-αβTCR heavy and light chains and the mutated H66 heavychains are set forth in Table 10. Mutated amino acids are shaded and theconsensus glycosylation target sites created by the mutation areunderlined.

TABLE 10 Amino acid sequences of H66 anti-αβTCR antibodies SEQ ID NOName Amino Acid Sequence 23 Anti-αβTCR cloneEIVLTQSPATLSLSPGERATLSCSATSSVSYMHWYQQ H66 light chainKPGQAPRRLIYDTSKLASGVPARFSGSGSGTSYTLTISSLEPEDFAVYYCQQWSSNPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYE KHKVYACEVTHQGLSSPVTKSFNRGEC* 24Anti-αβTCR clone EVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW H66 heavy chainVRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR DNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGFVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 25 Anti-αβTCR cloneEVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW H66 S298N/Y300SVRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR heavy chainDNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGFVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYN NTSRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 26 Anti-αβTCR cloneEVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW H66 S298N/VRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR T299A/Y300SDNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF heavy chainVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK* 27 Anti-αβTCR cloneEVQLLQSGGGLVQPGGSLRLSCAASGYKFTSYVMHW H66 N297Q/VRQAPGKGLEWVGYINPYNDVTKYNEKFKGRFTLSR S298N/Y300SDNSKNTLYLQMNSLRAEDTAVYYCARGSYYDYDGF heavy chainVYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAK TKPREEQYQNTSRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN HYTQKSLSLSPGK*

The mutant, wild-type, and two aglycosylated control (HEBE1 Agly IgG4and HEBE1 Δab IgG1 in pCEP4) constructs were transfected intoHEK293-EBNA cells in triple-flasks for expression. Proteins werepurified from 160 ml of conditioned media (CM) with 1 ml HiTrap proteinA columns (GE) using a multi-channel peristaltic pump. Five microgramsof each resulting supernatant were analyzed on 4-20% Tris-Glycinereducing and non-reducing SDS-PAGE gels (see FIG. 2). The heavy chainsof the aglycosylated mutants (N297Q, T299A, and Agly controls), havemigrated further (arrowhead), consistent with the loss of the glycans inthese antibodies. The heavy chains of the engineered glycosylatedantibodies (NSY, STY, SY, Δab, and wt control, arrows), however, migratesimilarly to the wild-type control. This result is consistent with theexistence of an engineered glycosylation site at EU position 298.SEC-HPLC analysis indicated that all mutants are expressed as monomers.

3B. Glycosylation Analysis by LC-MS

The engineered H66 IgG1 Fc variants were partially reduced with 20 mMDTT at 37° C. for 30 min. The samples were then analyzed by capillaryLC/MS on an Agilent 1100 capillary HPLC system coupled with a QSTAR qqTOF hybrid system (Applied Biosystems). A Bayesian proteinreconstruction with baseline correction and computer modeling in AnalystQS 1.1 (Applied Biosystem) was used for data analysis. In theS298N/T299A/Y300S H66 antibody mutant, one glycosylation site wasobserved at amino acid 298 with bi-antennary and tri-antennarycomplex-type glycans detected as the major species alongside G0F, G1Fand G2F (see FIG. 34). This altered glycosylation profile is consistentwhich shifted glycosylation at N298 instead of the wild-typeglycosylation site at N297.

3C. Binding Properties of αβTCR Antibody Mutants to Human FcγRIIIa andFcγRI Using Biacore

Biacore was used to assess binding to recombinant human FcγRIIIa (V158 &F158) and FcγRI. All four flowcells of a CMS chip were immobilized withanti-HPC4 antibody via the standard amine coupling procedure provided byBiacore. The anti-HPC4 antibody was diluted to 50 μg/mL in 10 mM sodiumacetate pH 5.0 for the coupling reaction and injected for 25 min at 5μL/min. Approximately 12,000 RU of antibody was immobilized to the chipsurface. Recombinant human FcγRIIIa-V158 and FcγRIIIa-F158 were dilutedto 0.6 μg/mL in binding buffer (HBS-P with 1 mM CaCl₂)) and injected toflowcells 2 and 4, respectively, for 3 min at 54/min to capture 300-400RU receptor on the anti-HPC4 chip. In order to distinguish between thelow binders, three times more rhFcγRIIIa was captured on the anti-HPC4surface than usually used in this assay. Flowcells 1 and 3 were used asreference controls. Each antibody was diluted to 200 nM in bindingbuffer and injected over all four flowcells for 4 min, followed by 5 mindissociation in buffer. The surfaces were regenerated with 10 mM EDTA inHBS-EP buffer for 3 min at 204/min. The results of these experiments areshown in FIG. 3.

Biacore was also used to compare the FcγRI binding. Anti-tetra Hisantibody was buffer exchanged into 10 mM sodium acetate pH 4.0 using aZeba Desalting column and diluted to 25 μg/mL in the acetate buffer foramine coupling. Two flowcells of a CMS chip were immobilized with 9000RU of the anti-Tetra-His antibody after 20 min injection at 54/min. Asin the previous experiment, ten times more FcγRI was captured to theanti-tetra-His surface in order to compare samples with weak binding.Recombinant human FcγRI was diluted 10 μg/mL in HBS-EP binding bufferand injected to flowcell 2 for 1 min at 54/min to capture 1000 RUreceptor to the anti-tetra-His chip. A single concentration of antibody,100 nM, was injected for 3 min at 304/min over the captured receptor andcontrol surface. Subsequently, dissociation was monitored for threeminutes. The surface was then regenerated with two 30 second injectionsof 10 mM glycine pH 2.5 at 204/min. The results of these experiments areshown in FIG. 4.

These results demonstrate a striking decrease in binding of theglycoengineered mutants to FcγRIIIa or FcγR1. H66 S298N/T299A/Y300S inparticular has almost completely abolished binding to both receptors.This mutant was chosen for more detailed analysis.

3D. Stability Characterization Using Circular Dichroism (CD)

The stability of the S298N/T299A/Y300S antibody mutant was monitored bya Far-UV CD thermo melting experiment in which the CD signal at 216 nmand 222 nm was monitored as increasing temperature lead to the unfoldingof the antibody (denaturation).

Temperature was controlled by a thermoelectric peltier (Jasco modelAWC100) and was increased at a rate of FC/min from 25-89° C. The CDspectra were collected on a Jasco 815 spectrophotometer at a proteinconcentration of approximately 0.5 mg/mL in PBS buffer in a quartzcuvette (Hellma, Inc) with a path length of 10 mm. The scanning speedwas 50 nm/min and a data pitch of 0.5 nm. A bandwidth of 2.5 nm was usedwith a sensitivity setting of medium. The CD signal and HT voltage werecollected from 210-260 nm with data intervals of 0.5 nm and attemperature intervals of 1° C. and four replicate scans were performedfor each sample. The results demonstrate that both delta AB H66 and theS298N/T299A/Y300S H66 mutant exhibit similar thermal behaviors and haveapproximately the same onset temperature for degradation (around 63 C)(FIG. 35), further suggesting that they have comparable stability.

Example 4: Functional Analysis of Fe-Engineered Mutants

Fc-engineered mutants were assessed through a PBMC proliferation assayand a cytokine release assay. In the PBMC proliferation assay, humanPBMC were cultured with increasing concentrations of therapeuticantibody for 72 hours, ³H-thymidine was added and cells were harvested18 hours later. For the T cell depletion/Cytokine Release assay, humanPBMC were cultured with increasing concentrations of therapeuticantibody and were analyzed daily for cell counts and viability (Vi-Cell,Beckman Coulter) out to day 7. Cell supernatants were also harvested,stored at −20° C. and analyzed on an 8-plex cytokine panel (Bio-Rad).

Normal donor PBMC were thawed and treated under the following conditions(all in media containing complement): Untreated; BMA031, moIgG2b 10ug/ml; OKT3, moIgG2a 10 ug/ml; H66, huIgG1 deltaAB 10 ug/ml, 1 μg/ml and0.1 ug/ml; H66, huIgG1 S298N/T299A/Y300S 10 ug/ml, 1 ug/ml and 0.1ug/ml.

Cytokines were harvested at day 2 (D2) and day 4 (D4) for BioplexAnalysis (IL2, IL4, IL6, IL8, IL10, GM-CSF, IFNg, TNFa). Cells werestained at D4 for CD4, CD8, CD25 and abTCR expression.

The results, shown in FIGS. 5-8, demonstrate that H66 S298N/T299A/Y300Sbehaved similarly to the H66 deltaAB in all the cell based assaysperformed, showing minimal T-cell activation by CD25 expression, bindingto abTCR (with slightly different kinetics to deltaAB), and minimalcytokine release at both D2 and D4 time points. The S298N/T299A/Y300Smutant thus eliminated effector function as effectively as the deltaABmutation.

Example 5: Preparation and Characterization of an Engineered Fc Variantin the Anti-CD52 Antibody Backbone

In addition to the H66 anti-αβTCR antibody, the S298N/Y300S mutation wasalso engineered in an anti-CD52 antibody backbone (clone 2C3). Thismutant was then examined in order to determine whether the observedeffector function modulation seen in the S298N/Y300S H66 anti-αTCRantibody was consistent in another antibody backbone.

5A. Creation of 2C3 Anti-CD52 Antibody Altered Glycosylation Variants

First, S298N/Y300S 2C3 variant DNA was prepared by quick changemutagenesis using pENTR_LIC_IgG1, and WT 2C3 VH was cloned into themutated vector by LIC. Full-length mutants were cloned into the pCEP4(-E+I)Dest expression vector using Gateway technology. Mutations weresubsequently confirmed by DNA sequencing and the sequences are set forthin Table 11. Mutated amino acids are shaded and the consensusglycosylation target sites created by the mutation are underlined. Themutants were then transfected into HEK293-EBNA cells in a 6-well plateformat and the protein was purified from conditioned media. Anti-CD522C3 wild-type antibody was produced in parallel as a control. Theexpression level was found to be 0.1 μg/mL using SD-PAGE and Westernblot analyses (FIG. 9A). Expression of mutants in neat conditioned mediawas also measured by protein A capture on Biacore. Concentration wasdetermined using the dissociation response after a six-minute injectionto immobilized protein A. CHO-produced WT 2C3 serially diluted in mediafrom 90 μg/mL to 1.5 ng/mL was used as a standard curve. Concentrationswere calculated within approximately 0.2 μg/mL by a calibration curveusing a 4-parameter fit. Relative expression levels were low andgenerally agree with the Western blot data (FIG. 9B).

TABLE 11 Anti-CD52 clone 2C3 antibody sequences SEQ ID NO NameAmino Acid Sequence 28 Anti-CD-52DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLNWL 2C3 WTLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISR light chainVEAEDVGVYYCVQGTHLHTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVT HQGLSSPVTKSFNRGEC* 29Anti-CD-52 EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNWVR 2C3 WTQAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDS heavy chainKNSLYLQMNSLKTEDTAVYYCTPVDFWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK* 30 Anti-CD-52EVQLVESGGGLVQPGGSLRLSCAASGFTFNTYWMNWVR 2C3QAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDS S298N/Y300SKNSLYLQMNSLKTEDTAVYYCTPVDFWGQGTTVTVSSAS heavy chainTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNNTSRVVSVLTVLHQDWLNGKE YKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK*

5B. Glycosylation Analysis Using PNGaseF

To evaluate the additional glycosylation sites introduced by themutation, the enriched S298N/Y300S mutant was de-glycosylated withPNGase F. Deglycosylation did not demonstrate any apparent change inmolecular weight, which indicates that no additional carbohydrate waspresent (FIG. 10). Small scale preparations were performed in order topurify these mutants for further characterization and the resultsreconfirmed that there was not an additional carbohydrate present on theS298N/Y300S mutant (FIG. 11).

5C. Binding Properties of 2C3 Anti-CD52 Antibody Mutants to HumanFcγRIIIa Using Biacore

Biacore was also used to characterize the antigen-binding, FcγRIII, andbinding properties of the purified antibodies (see FIGS. 12, 13, and14). The S298N/Y300S 2C3 variant bound to the CD52 peptide tightly andthe binding sensorgram was undistinguishable from the wild-type control,demonstrating that this mutation does not affect its antigen binding(FIG. 12A).

To assay for Fc effector function, FcγRIII receptor (Val158) was used inbinding studies. The mutant and wild-type control antibody were dilutedto 200 nM and injected to HPC4-tag captured FcγRIIIa. FcγRIII bindingwas almost undetectable for the S298N/Y300S mutant, which indicated aloss of effector function by this variant (FIG. 12B and FIG. 14A). Tofurther assay for Fc effector function, the FcγRIII receptor (Phe158)was also used in binding studies. The mutant and wild-type controlantibodies were diluted to 200 nM and injected to HPC4-tag capturedFcγRIIIa. FcγRIII binding was almost undetectable for the S298N/Y300Smutant, which indicates a loss of effector function with the Phe158variant (FIG. 14B). Finally, Biacore was used to compare the FcRnbinding properties of the purified proteins. Mouse and SEC-purifiedhuman FcRn-HPC4 were immobilized to a CMS chip via amine coupling. Eachantibody was diluted to 200, 50, and 10 nM and injected over thereceptors. Campath, CHO-produced WT 2C3, and DEPC-treated Campath wereincluded as positive and negative controls. These data show that themutant binds to both human and murine FcRn receptor with the sameaffinity as the wild-type antibody control and that it likely has noalterations in its circulation half-life or other pharmacokineticproperties. (see FIG. 12C, FIGS. 13A and B). Accordingly, theS298N/Y300S mutation is applicable to antibodies in general, to reduceor eliminate undesired Fc effector function, for example throughengagement of human Fcγ receptors.

Example 6: Circulating Immune Complex Detection in the S298N/Y300SMutant

Circulating immune complex detection was also investigated using a C1qbinding assay for the S298N/Y300S mutant and WT control. High bindingCostar 96-well plates were coated overnight at 4° C. with 100 μl of2-fold serially diluted 2C3 Abs at concentrations ranging from 10-0.001μg/ml in coating buffer (0.1M NaCHO₃ pH 9.2). ELISA analysis showed thatC1q binding is reduced for the S298N/Y300S mutant compared to WT (FIG.15A). The binding of anti-Fab Ab to the coated 2C3 Abs confirmedequivalent coating of the wells (FIG. 15B).

Example 7: Separation and Analysis of S298N/Y300S Mutant UsingIsoelectric Focusing

A pH 3-10 Isoelectric Focusing (IEF) gel was run to characterize theS298N/Y300S mutants. S298/Y300S was found to have more negative charges,and therefore, likely more sialic acid molecules (FIG. 18A). Both theS298N/Y300S mutant and WT 2C3 were shown by intact MS to have G0F andG1F as the dominant glycosylation species (FIGS. 18 B and D,respectively).

Example 8: Antigen Binding Affinity of S298N/Y300S

Biacore was used to compare the antigen binding affinity of WT anti-CD522C3 Ab and the S298N/Y300S mutant that had been prepared and purifiedfrom both smaller (FIG. 16) and larger (FIG. 17) scale expressions. CMSchips immobilized with CD52 peptide 741 and control peptide 777 wereobtained. Antibodies were serially diluted 2-fold from 60 to 0.2 nM inHBS-EP and were then injected over the chip surface for 3 min followedby a 5 min dissociation in buffer at a flow rate of 50 μl/min. Thesurface was then regenerated with a pulse of 40 mM HCl. These analyseswere performed in duplicate and demonstrate that the S298N/Y300S mutantand WT 2C3 antibodies show comparable CD52 peptide binding.

A media screening platform was designed to test functional bindingproperties prior to purification in order to screen antibodies createdduring small scale transfections. These tests were performed using Octet(FIG. 19A) to determine concentration and used Protein A biosensors anda GLD52 standard curve. Samples were diluted to 7.5 and 2 nM in HBS-Epfor a CD52 binding comparison using Biacore (FIG. 19B). The results ofthe peptide binding assay showed that both the S298N/Y300S mutant andthe WT 2C3 antibodies have comparable CD52 peptide binding. Furthermore,these analyses demonstrate that Octet and Biacore work well to predictantigen binding by antibodies from small scale transfections.

Example 9: Preparation of S298N/Y300S, S298N/T299A/Y300S, andN297Q/S298N/Y300S Altered Glycosylation Mutants in Additional AntibodyBackbones

In addition to the anti-αβ-TCR antibody and 2C3 anti-CD-52 antibody, theS298/Y300S, S298N/T299A/Y300S, and N297Q/S298N/Y300S mutations wereengineered in other antibody backbones to confirm that the additionaltandem glycosylation site could be introduced into unrelated heavy chainvariable domain sequences. The alternatively glycosylated anti-CD-5212G6 and anti-Her2 mutants are set forth in Tables 12 and 13. Mutatedamino acids are shaded and the consensus glycosylation target sitescreated by the mutation are underlined.

TABLE 12 Anti-CD52 clone 12G6 antibody sequences SEQ ID NO NameAmino Acid Sequence 31 Anti-CD-52DIVMTQTPLSLSVTPGQPASISCKSSQSLLYSNGKTYLNWV 12G6 WTLQKPGQSPQRLIYLVSKLDSGVPDRFSGSGSGTDFTLKISRV light chainEAEDVGVYYCVQGSHFHTFGQGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGEC 32 Anti-CD-52EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ 12G6 WTAPGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN heavy chainSLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK* 33 Anti-CD-52EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ 12G6APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN S298N/Y300SSLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG heavy chainPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV HNAKTKPREEQYNNTSRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK* 34 Anti-CD-52EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ 12G6 S298N/APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN T299A/Y300SSLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG heavy chainPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK* 35 Anti-CD-52EVQLVESGGGLVQPGGSLRLSCAASGFPFSNYWMNWVRQ 12G6 N297Q/APGKGLEWVGQIRLKSNNYATHYAESVKGRFTISRDDSKN S298N/Y300SSLYLQMNSLKTEDTAVYYCTPIDYWGQGTTVTVSSASTKG heavy chainPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYQNTSRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GK*

TABLE 13 Anti-Her2 antibody sequences SEQ ID NO Name Amino Acid Sequence36 Anti-Her2 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKP WTGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDF light chainATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPV TKSFNRGEC* 37 Anti-Her2EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA WTPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY heavy chainLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK* 38 Anti-Her2EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQA S298N/T299A/PGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAY Y300SLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTV heavy chainSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNNASRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK*

Example 10. Generation of Altered Antibodies Containing Reactive GlycanMoieties

In order to generate antibodies containing glycan moieties capable ofreacting with derivatized effector moieties, an anti-HER antibody wasfirst glycosylated in vitro using glycosyltransferase and relevant sugarnucleotide donors. For example, to introduce the sialic acid residues,donor antibodies were first galactosylated with β-galactosyltransferase,followed with sialylation with α2,6-sialyltransferase according to themethods of Kaneko et al. (Kaneko, Y., Nimmerjahn, F., and Ravetch, J. V.(2006) Anti-inflammatory activity of immunoglobulin G resulting from Fcsialylation. Science 313, 670-3). The reaction was performed in aone-pot synthesis step using β-galactosyltransferase (50 mU/mg, Sigma)and α2,6-sialyltranafrease (5 ug/mg, R&D system) with donor sugarnucleotide substrates, UDP-galactose (10 mM) and CMP-sialic acid (10 mM)in 50 mM MES buffer (pH 6.5) containing 5 mM MnCl₂. The reaction mixturecontaining 5 mg/ml anti-HER2 antibody was incubated for 48 hours at 37°C. The sialylation was verified using MALDI-TOF MS analysis ofpermethylated glycans released from the antibody with PNGase F, sialicacid content analysis using Dionex HPLC and lectin blotting with SNA, alectin specific for α2,6-sialic acid.

MALDI-TOF analysis of glycans released by PNGase F treatment of thesialylated anti-HER2 antibody indicated that native glycans had beencompletely remodeled with a mainly monosialylated biantennary structure,A1F (FIG. 27A) together with small amount of disialylated species.Treatment of the antibody with higher amounts of α2,6-sialyltransferaseproduced more homogenous populations of the A1F glycoform, suggestingthat either the enzyme activity or glycan localization may prevent fullsialylation. Sialic acid content was determined to be ˜2 mol per mol ofantibody, which is consistent with A1F glycan as the major glycoformspecies (FIG. 27B). Lectin blotting with a SAN lectin, Sambucus nigraagglutinin specific for α2,6-linked sialic acid, confirmed that thesialic acid was present in an α2,6-linkage configuration (FIG. 27C).

In conclusion, although the native protein glycans are somewhatheterogeneous, remodeling through galactosyl and sialyltransferasesyields a nearly homogeneous antibody with monosialylated but fullygalactosylated biantennary glycans (A1F). The introduction of only ˜1sialic acid on the two galactose acceptors on each branched glycan maybe due to limited accessibility of one of the galactoses from glycanswhich are often buried in the antibody or non-covalent interactions ofthe glycans with the protein surface.

Example 11. Oxidation of Altered Antibodies Containing Reactive GlycanMoieties

Once the sialylation was verified, the in-process oxidation ofsialylated anti-HER2 antibody with various concentrations of periodate(0.25 to 2 mM) was investigated. The sialylated antibody was firstbuffer-exchanged into 25 mM Tris-HCl (pH 7.5) containing 5 mM EDTAfollowed by buffer exchange with PBS buffer. The buffered antibodymixture was then applied to protein A Sepharose column pre-equilibratedwith PBS buffer. After the column was washed with 15 column volumes ofPBS, 15 column volumes of PBS containing 5 mM EDTA, and 30 columnvolumes of PBS, it was then eluted with 25 mM citrate phosphate buffer(pH 2.9). The eluates were immediately neutralized with dibasicphosphate buffer and the antibody concentrated using Amicon ultra fromMillipore. Following purification, the sialylated anti-HER2 antibodythen was oxidized with sodium periodate (Sigma) in 100 mM sodium acetatebuffer (pH 5.6) on ice in the dark for 30 minutes, and the reactionquenched with 3% glycerol on ice for 15 minutes. The product wasdesalted and exchanged into 100 mM sodium acetate (pH 5.6) by 5 roundsof ultrafiltration over 50 kDa Amicons. FIG. 28A shows sialic acidcontent analysis of sialylated antibody titrated with various amounts ofperiodate. Complete oxidation of the sialic acid residues was achievedat a periodate concentration above 0.5 mM. Indeed, a periodateconcentration as low as 0.5 mM was enough to fully oxidize theintroduced sialic acid. Accordingly, a 1 mM concentration of periodatewas chosen for oxidation of sialylated antibody for drug conjugation.

Oxidation can have adverse effects on the integrity of an antibody. Forexample, the oxidation of methionine residues, including Met-252 andMet-428, located in Fc CH3 region, close to FcRn binding site is knownto affect FcRn binding which is critical for prolonging antibody serumhalf-life (Wang, W., et al. (2011) Impact of methionine oxidation inhuman IgG1 Fc on serum half-life of monoclonal antibodies. Mol Immunol48, 860-6). Accordingly, to examine the potential side effects ofperiodate oxidation on methionine residues (e.g., Met-252) critical forFcRn interaction, the oxidation state of the sialylated antibody wasdetermined by LC/MS analysis of a trypsin peptide digest. This analysisrevealed ˜30% oxidation of Met-252 and <10% oxidation of Met-428 aftertreatment of the sialylated trastuzumab with 1 mM periodate. Todetermine the impact of this degree of methionine oxidation on FcRnbinding, the FcRn binding kinetics for each antibody was evaluated usingsurface plasmon resonance (BIACORE). This analysis revealed thatoxidation state correlated with a minor loss in FcRn binding (12% and26% reduction to mouse and human FcRn, see FIGS. 28B and 28Crespectively). Notably, a ˜25% reduction in the Ka for human FcRn hasbeen reported to have no effect on the serum half-life in a human FcRntransgenic mouse, since a single intact FcRn site on each antibody issufficient to provide functionality and the PK advantage (Wang et al.,Id).

In summary, these data indicate that the introduction ofperiodate-sensitive sialic acid residues by sialyltransferase treatmentpermits the use of much lower concentrations of periodate, resulting inminimal side effects on antibody-FcRn interactions and antibodyintegrity as assessed by aggregation (1%). Thus, the use of sialylatedantibodies provides a wider window of oxidation conditions to beemployed, allowing the reproducible generation of active glycoconjugateswithout an effect on serum half-life.

The galactose in a hyperglycosylated antibody mutant can also beoxidized specifically using galactose oxidase to generate an aldehydegroup for conjugation. To confirm this approach, an A114N anti-TEM1antibody was concentrated to 13-20 mg/ml and then treated with 20 mU/mgsialidase in PBS for 6 hours at 37° C. The desialated product was thenoxidized with galactose oxidase (“GAO”), first with 5 ug GAO/mg proteinovernight at 37° C. followed by addition of 2 ug GAO/mg protein andincubation for an additional 5 hours. Sodium acetate was added to adjustthe pH to 5.6 (0.1 v/v, pH5.6), and DMSO was added to achieve a finalreaction concentration of 16%, were added prior to conjugation. Thehyperglycosylation mutant A114N anti-HER antibody (15 mg/ml) wassimilarly desialylated with sialidase (20 mU/mg) and oxidized with 5 ugGAO per mg protein in a single reaction overnight at 37° C.

Example 12. Synthesis of Reactive Effector Moieties

In order to facilitate conjugation with aldehyde-derivatized antibodyglycoforms, candidate drug effector moieties (e.g., MomomethylAuristatin E (MMAE) and Dolastatin 10 (Dol10)) were derivatized withaminooxy-cystamide to contain functional groups (e.g., aminooxy-cys)specifically reactive with the aldehyde.

Briefly, to generate aminooxy-cystamide as a starting material,S-Trityl-L-cysteinamide (362 mg, 1 mmol) was added to a 3 mL of a DMFsolution of t-BOC-aminooxyacetic acid N-hydroxysuccinimide ester (289mg, 1 mmol). The reaction was complete after 3 h as evident from HPLCanalysis. The reaction mixture was subsequently diluted with 30 ml ofdichloromethane and was washed with 0.1 M sodium bicarbonate solution(2×20 mL), water (2×20 mL), and brine (2×20 mL). The solution was driedover anhydrous sodium sulfate, filtered and concentrated to dryness. Tothis dried residue was added 3 mL of TFA followed by 150 μL oftriethylsilane. The resulting solution was precipitated from t-butylmethyl ether and the process repeated three times. After filtration, theresidue was dried under reduced pressure yielding 205 mg of an off whitesolid (67% yield). The compound was used for next step without furtherpurification.

To generate aminooxy-derivatized MMAE (Aminooxy-Cys-MC-VC-PABC-MMAE),30.1 mg of aminooxy-cystamide (0.098 mmol, 2 eq.) was combined with 64.6mg of MC-VC-PABC-MMAE (0.049 mmol), and 100 μL of triethylamine in 3 mLof DMF. The resulting reaction mixture was stirred at room temperaturefor 15 minutes, by which time reaction was complete according to HPLCanalysis. The compound was purified by preparative HPLC yielding 45 mg(62%) of the desired product as an off-white solid. Reversed-phase HPLCanalysis suggested the purity of the compound to be >96%. ESI calcd forC73H116N14018S (MH)⁺ 1509.8501; found, m/z 1509.8469.

To generate aminooxy-derivatized Dol10(Aminooxy-Cys-MC-VC-PABC-PEG8-Dol10), 7.4 mg (0.024 mmol, 3 eq.) ofaminooxy-cystamide, 12 mg (0.008 mmol) of MC-VC-PABC-PEG8-Dol10 and 30μL triethylamine were combined in in 3 mL of DMF. The reaction wascomplete within 15 minutes according to HPLC analysis. Preparative HPLCpurification resulted in 6.2 mg (46%) of the desired product as anoffwhite solid. Reversed-phase HPLC analysis suggests the purity of thecompound to be >96%. ESI calcd for C80H124N16019S2 (MH)⁺ 1678.0664;found, m/z 1678.0613.

Example 13. Sialic Acid-Mediated (SAM) Conjugation of Reactive EffectorMoieties

Following desalting, drug-linkers of Example 11 were combined with theoxidized, sialylated antibodies of Example 10 with 75% DMSO (0.167 v/v)at a concentration of 25 mM to achieve a 24:1 molar ratio of drug-linkerto antibody and a final antibody concentration at 5 mg/ml. The mixturewas incubated overnight at room temperature. The unincorporateddrug-linkers and any free drugs were scavenged using BioBeads. Theproduct was buffer-exchanged into Histidine-Tween buffer using PD-10columns and sterile filtered. The endotoxin levels were determined andless than 0.1 EU/mg ADC was achieved for in vivo study.

FIG. 29A-C shows a hydrophobic interaction chromatograph (HIC) ofdifferent sialylated antibodies (anti FAP B11 and G11 and the anti-HER2antibody of Example 11) glycoconjugated to AO-MMAE. Sialylated HER2antibody was also conjugated with the drug-linker,AO-Cys-MC-VC-PABC-PEG8-Dol10 (FIG. 29D). This analysis reveals thatthere are mainly one or two drug conjugates per antibody with adrug-to-antibody ratio (DAR) ranging from 1.3-1.9. The increasedretention time of the Dol10 glycoconjugate (FIG. 29D) as compared to theMMAE glycoconjugate (FIG. 29C) is likely due to the greaterhydrophobicity of Dol10.

LC-MS analysis was also conducted with an anti-HER antibody conjugatedwith two different drug-linkers (AO-MMAE or AO-PEG8-Dol10) at 30 mgscale. This analysis showed similar DAR values of 1.7 and 1.5 followingconjugation, which is comparable to HIC analysis. Size-exclusionchromatography (SEC) showed very low levels (1%) of aggregates in theseconjugates.

Example 14. Galactose-Mediated (GAM) Conjugation of Reactive EffectorMoieties

The galactose aldehyde generated with galactose oxidase on the A114NantiTEM1 hyperglycosylation mutant antibody as described in Example 11was conjugated with 24 molar excess of aminooxy-MC-VC-PABC-MMAEdrug-linker over antibody by overnight incubation at 25° C., yielding aADC conjugate with a DAR of 1.72.

To the galactose oxidase-treated antiHER antibody prepared as describedin Example 11, one tenth reaction volume of 1M sodium acetate, pH5.6,was added to adjust the pH to 5.6 and DMSO was added to make the finalconcentration of 14% before adding 24 eq. aminooxy MC-VC-PABC-MMAE druglinker. The reactions were incubated for overnight at room temperature.Free drug and drug-linker were scavenged with Biobeads and the productbuffer exchanged by SEC (65% yield). The product conjugate was analyzedby HIC. As shown in FIG. 30, AO-MMAE had been conjugated to ˜60% of themolecules.

Example 15. In Vitro ADC Cell Proliferation Assays

The in vitro activity of the anti-HER and anti-FAP glycoconjugatemolecules were also compared with corresponding thiol conjugatescontaining the same drug moiety linked via thiol linkages to hingeregion cysteines of the same donor antibody. The thiol conjugatescontained approximately twice the number of drugs per antibody (DAR)than the glycoconjugates. Thiol-based conjugation was performed asdescribed by Stefano et al (Methods in Molecular Biology 2013, inpress). Her2+SK-BR-3 and Her2-MDA-MB-231 cell lines were then employedto evaluate the relative efficacy of each ADC. The results of thisanalysis are presented in Table 15 below

TABLE 15 EC₅₀ comparison of glycoconjugates and thiol conjugates EC₅₀DAR (ng/ml) Anti-HER-MC-VC-PABC-MMAE 3.8* 2.3 (Thiol MMAE)AntiHER-AO-Cys-MC-VC-PABC-MMAE 1.7* 4.7 (Glyco MMAE)Anti-HER-MC-VC-PABC-PEG8-Dol10 3.9* 0.45 (Thiol Dol10)Anti-HER-AO-Cys-MC-VC-PABC- 1.5* 0.97 PEG8-Dol10 (Glyco Dol10)Anti FAP B11-MC-VC-PABC-MMAE 3.3** 382.4 (Thiol MMAE), CHO + FAPAnti FAP B11-AO-Cys-MC-VC-PABC- MMAE (Glyco MMAE), CHO + FAP 1.5** 682.4Note: *DAR determined by LC-MS; **DAR determined by HIC

FIGS. 31(A-D) show a comparison of in vitro potency of anti-HERglycoconjugate and its counterpart thiol conjugate. Cell viability wasdetermined following 72 hr exposure of the conjugates to Her2 antigenexpressing (SK-BR-3) cells (FIGS. 31A and C) or non-expressing(MDA-MB-231) cells (FIGS. 31B and D). The ADCs contained either MMAE orPEGS-Dol10 linked to the glycans (“glyco”) or by conventional chemistryto hinge region cysteines (“thiol”). As shown in FIGS. 31A and C,˜2-fold lower EC₅₀ was observed for the thiol conjugates compared to theglycoconjugates, which is consistent with 2-fold higher DAR in theformer than the latter. No toxicity was observed with the Her2-cell linewith any antibody up to 100 ug/ml.

Similar trends were also observed in the cell proliferation for ADCprepared with antibodies against a tumor antigen (FAP) which is highlyexpressed by reactive stromal fibroblasts in epithelial cancersincluding colon, pancreatic and breast cancer (Teicher, B. A. (2009)Antibody-drug conjugate targets. Curr Cancer Drug Targets 9, 982-1004).These conjugates were again prepared by conjugating either aminooxy MMAEdrug-linker or maleimido MMAE drug-linker to glycans or a thiol group.Cell proliferation assays of these conjugates showed that EC₅₀ of thethiol conjugate had ˜100-fold higher potency on the CHO cellstransfected with human FAP than the same cells lacking FAP expression asdepicted in FIG. 32, which shows a comparison of in vitro potency ofanti FAP B11 glycoconjugate and thiol conjugate. Cell viability wasdetermined following exposure of the conjugates to CHO cells transfectedwith or without FAP antigen. The ADCs contained MMAE linked to theglycans (“glyco”) or by conventional chemistry to hinge region cysteines(“thiol”). Note that the ˜2-fold lower EC50 for the thiol compared tothe glycoconjugates is consistent with the relative amounts of drugdelivered per antibody assuming similar efficiencies for target bindingand internalization in antigen expressing CHO cells. In parallel, aglycoconjugate of anti FAP (B11) ADC with a DAR of 1.5 as describedpreviously was assayed and showed an ˜2-fold higher EC₅₀ than comparatorthiol conjugate (DAR 3.3).

As shown in FIG. 36, similar trends were observed in the cellproliferation assay for ADC prepared with the anti-HER antibody bearingthe A114N hyperglycosylation mutation and AO-MMAE as described inExample 14, when assayed on SK-BR-3 expressing cells or MDA-MB-231cells. The A114N glycoconjugate clearly shows enhanced cell toxicityagainst the Her2 expressing cell line over the non-expressing line. Therelative toxicity compared to the SialT glycoconjugate prepared with thesame antibody is consistent with the lower drug loading of thispreparation.

A cell proliferation assay was also performed for ADC prepared with theanti-TEM1 antibody bearing the A114N hyperglycosylation mutation andAO-MMAE prepared as described in Example 14. Higher toxicity wasobserved with the TEM1-expressing cells lines SJSA-1 and A673 comparedto the non-expressing MDA-MB-231 line. The level of toxicity comparedwith a conventional thiol conjugate with the same antibody was inkeeping with the drug loading (DAR) of this preparation.

SJSA-1 A673-RPMI A673-DMEM-RPMI MDA-MB-231 IC50 IC50 IC50 IC50 antiTEM1A114N-AO-MC-VC- 3 μg/ml 3.2 μg/ml 2.2 μg/ml 40 μg/ml PABC-MMAEantiTEM1-MC-VC-PABC-MMAE 4 μg/ml 1 μg/ml 0.9 μg/ml 20 μg/ml

In summary, the site-specific conjugation of the drugs through theglycans with cleavable linkers produces ADCs with toxicities and invitro efficacy that are equivalent to conventional thiol-basedconjugates, as demonstrated using different antibodies and differentdrug-linkers. Moreover, below 2 mM periodate, the level of drugconjugation correlates with the reduction of sialic acid. Increasingperiodate concentration above 2 mM produces little benefit, as expectedfrom the complete conversion of sialic acid to the oxidized form.However, under all conditions, the number of drugs per antibody wasslightly lower than the sialic acid content, indicating that some of theoxidized sialic acids may similarly not be available for coupling,either because of being buried or otherwise due to steric hindrancearising from the bulk of the drug-linker.

Example 16. In Vivo Characterization of Antibody Drug Conjugates

Efficacy of anti-HER glycoconjugates were also evaluated in a Her2+tumor cell xenograft mode and compared with thiol conjugate comparatorshaving ˜2-fold higher DAR. Beige/SCID mice were implanted with SK-OV-3Her2+ tumor cells which were allowed to establish tumors of ˜150 mm³prior to initiation of treatment. ADCs at 3 or 10 mg/kg doses wereinjected through tail vein on days 38, 45, 52 and 59. There were ˜10mice per group. The tumor volume of mice in different group was measuredand their survival was recorded. The survival curve was plotted based onKaplan-Meier method.

FIGS. 33(A-D) show a comparison of in vivo efficacy of the anti-HERglycoconjugates and thiol conjugates in a Her2+ tumor cell xenograftmodel. Beige/SCID mice implanted with SK-OV-3 Her2+ tumor cells weredosed with MMAE (FIGS. 33 A and B) and PEGS-Dol10 (FIGS. 33 C and D)containing glycoconjugates or a thiol conjugate comparators with ˜2-foldhigher DAR. The tumor growth kinetics of the MMAE conjugates is shown inFIG. 33A. In this case, the glycoconjugate showed a significantly higherefficacy than the naked antibody alone (black) but less than a thiolconjugate comparator having a ˜2-fold higher DAR (green). The MMAEglycoconjugate showed significant tumor regression and a ˜20 day delayin tumor growth (FIG. 33A) and ˜2-fold increase in survival time fromfirst dose (FIG. 33B). The thiol MMAE conjugate showed near-completetumor suppression at the same dose of ADC (10 mg/kg).

The in vivo efficacy of a PEG8-Dol10 glycoconjugate (“Glyco Dol10”) anda thiol conjugate comparator with ˜2-fold higher DAR (“Thiol Dol10”) wasalso determined in the same Her2+ tumor cell xenograft model. Bothconjugates showed lower efficacy than MMAE conjugates as describedpreviously. However, the aminooxy-PEG8-Dol10 glycoconjugate (“GlycoDol10”) at 10 mg/kg showed a 15-day delay in tumor growth (FIG. 33C) and˜20 day (1.7-fold) increase in survival time following firstadministration (FIG. 33D). The thiol conjugate was more efficacious atthe same dose, showing a 2-fold increase in survival. At a lower dose (3mg/kg), the thiol conjugate showed a lesser efficacy than theglycoconjugate at 10 mg/kg. This dose corresponds to 80 umol PEG8-Dol10drug per kg dose, compared to 110 umol PEG8-Dol10 drug per kg dose forthe glycoconjugate.

These data demonstrate that site-specific conjugation of drugs ontosialic acid of antibody glycans yields molecules with comparable potencyas ADCs generated via thiol-based chemistry. The somewhat lower in vivoefficacy likely stems from the fewer number of drugs which are carriedby each antibody into the tumor cells by the internalization of eachantibody-bound antigen. Although we have not compared theseglycoconjugates with thiol conjugates of the same DAR, the efficacyobserved at different doses of the two ADCs representing comparablelevels of administered drug shows that the glycoconjugates havecomparable intrinsic efficacy as their thiol counterparts, indicating nodeleterious effect of conjugation at this site. Moreover, a 10 mg/kgdose of the Dol10 glycoconjugate which introduced only 28% more drugprovided a 2-fold increase in survival over the thiol conjugate (at 3mg/kg), suggesting these conjugates may even provide superior efficaciesat the same DAR. Given the apparent limitation in sialic acidincorporation at native glycans, higher drug loading could be achievedby a number of different strategies including the use of branched druglinkers or the introduction of additional glycosylation sites and usingthe same method.

Example 17. Conjugation of Targeting Moieties

FIG. 37 demonstrates the overall scheme for the conjugation of targetingmoieties to existing carbohydrates or engineered glycosylation sites.This conjugation can be performed through the attachment of neoglycans,glycopeptides, or other targeting moieties to oxidized sialylatedantibodies (FIGS. 38 and 39). Moieties suitable for conjugation mayinclude those containing aminooxy linkers (FIGS. 40 and 41).

Example 18. Conjugation Through Sialic Acid in Native Fc Glycans

Man-6-P hexamannose aminooxy was conjugated to either a polyclonalantibody or monoclonal antibody specifically targeting a Man-6-Preceptor. The SDS-PAGE and MALDI-TOF MS analyses of the conjugation ofthe rabbit polyclonal antibody with Man-6-P hexamannose aminooxy isshown in FIG. 42. FIG. 43 depicts the results of surface plasmonresonance experiments used to assess the binding of control and Man-6-Phexamannose conjugated rabbit polyclonal IgG antibodies tocation-independent Man-6-P receptor (CI-MPR). In vitro analyses of thisconjugated antibody demonstrates increased uptake into both HepG2 (Homosapiens liver hepatocellular carcinoma) and RAW (Mus musculus murineleukemia) cell lines (FIG. 44). Cultures were stained withanti-rabbit-Alexa 488 antibody counterstained with DAPI.

Antibodies conjugated with Man-6-P or lactose aminooxy moieties werefurther tested through SDS-PAGE and lectin blotting and compared withunconjugated antibodies (FIG. 45). MALDI-TOF intact protein analyses ofthe control and conjugated antibodies demonstrate that the conjugateshave approximately two glycan moieties per antibody, while controlantibodies have none (FIG. 46).

Example 19. Conjugation Through Sialic Acid to Hinge Cysteine Residuesin Antibody

Man-6-P hexamannose maleimide (FIG. 41) was conjugated to either apolyclonal antibody or monoclonal antibody.

The conjugation of a polyclonal antibody with Man-6-P hexamannosemaleimide through hinge cysteines was examined through SDS-PAGE, lectinblotting, and Man-6-P quantitation (to determine the number of glycansconjugated per antibody) (FIG. 47). Conjugation of a polyclonal antibodywith lactose maleimide was also examined through the use of SDS-PAGE andgalacose quantitation of the control antibody, conjugated antibody, andfiltrate are shown in FIG. 48. Little increased aggregation was observedin hinge cysteine-conjugated polyclonal antibodies by size exclusionchromatography (SEC) (FIG. 50).

The conjugation of a monoclonal antibody with Man-6-P hexamannosemaleimide through hinge cysteines was also examined through SDS-PAGE andglycan quantitation (to determine the number of glycans conjugated perantibody) (FIG. 49). Little increased aggregation was observed in hingecysteine-conjugated polyclonal antibodies by size exclusionchromatography (SEC) (FIG. 51).

The binding of Man-6-P receptor (CI-MPR) to bis Man-6-P hexamannoseconjugated polyclonal and monoclonal antibodies through native Fcglycans or hinge disulfides was also demonstrated through gel shift on anative PAGE (FIG. 55).

Example 20. Preparation of Fully Galactosylated Monoclonal Antibody andConjugation of a Trigalactosylated Glycopeptide to the SialylatedAntibody

A mouse monoclonal antibody mutant with an STY mutation (NNAS) wasmodified with sialidase and galactosyltransferase for making mainlynative trigalactosylated glycans (2 glycans per antibody). The samemutant was also sialylated with sialyltransferase and conjugated with atrivalent galactose containing glycopeptide (FIG. 68) using SAMapproach. The sialic acid content of the enzyme modified antibodies wasexamined (FIG. 52). Further, MALDI-TOF analysis of the glycans releasedfrom control and desialylated/galactosylated (FIG. 53) NNAS as well asthe glycans released from control and sialylated (FIG. 54) NNAS wereexamined. SDS-PAGE (4-12% NuPAGE) and lectin blotting of enzyme modifiedand conjugated NNAS are shown in (FIG. 56). Terminal galactosequantitation was also measured for the control NNAS antibody,desialylated/galactosylated NNAS antibody, and glycopeptide conjugatedNNAS antibody (FIG. 57).

Example 21. Preparation of α2,3 Sialylated Lactose Maleimide Using aChemoenzyme Approach and Subsequent Conjugation to Non-Immune Rabbit IgGThrough Hinge Disulfides

Carbohydrate-binding proteins (including Siglec proteins) are able tobind more efficiently to areas with greater sialic acid density. Thus,the monosialylated glycans on a given antibody may not provide enoughsialic acid density to facilitate binding to other Siglec proteins.Therefore, a hinge disulfide conjugation approach for introducingmultiple copies of sialylated glycans was investigated. To producesialylated glycans for conjugation, lactose maleimide (5 mg) wassialylated in vitro with α2,3 sialyltransferase from Photobacteriumdamsela in Tris buffer (pH 7.5) for 2 hrs at 37° C. A control glycan wasincubated without sialyltransferase and compared with the originalglycans. MALDI-TOF MS analysis showed that the incubation of lactosemaleimide without enzyme in Tris buffer (pH 7.5) for 2 hrs at 37° C. didnot change the expected molecular weight of the molecule, suggestingthat the examined condition did not result in maleimide hydrolysis. TheMALDI-TOF and Dionex HPLC analysis of glycans modified with α2,3sialyltransferase indicate the presence of sialyllactose, although notas major peak (data not shown). Therefore, the sialyllactose maleimidewas additionally purified using QAE-sepharose columns and each fractionwas subsequently analyzed using MALDI-TOF and Dionex HPLC. Theseanalyses indicated that sialyllactose maleimide existed as major speciesin the 20 mM NaCl eluate from QAE column (FIG. 58). The amount ofsialylated glycans purified was estimated using sialic acid quantitationanalysis of the samples, indicating a recovery of ˜1.8 mg sialyllactosemaleimide.

Subsequent conjugation of a rabbit polyclonal antibody with thissialyllactose maleimide was tested using thiol-maleimide chemistry. Arabbit IgG antibody (1 mg) was reduced with TCEP at a 4 molar excess(over the antibody) for 2 hrs at 37° C. before being conjugated to 24molar excess of sialyllactose for 1 hr at room temperature. Theconjugate was then buffer-exchanged into PBS for analysis on SDS-PAGE(FIG. 59A). Sialic acid quantitation was also performed using DionexHPLC (FIG. 59B). Aliquots of control and thiol conjugate were treatedwith or without sialidase (1 U per mg) overnight at 37° C. beforesupernatants were recovered through filtration (10 kDa MWCO). The sialicacid content of the supernatants was measured and compared to samplestreated without sialidase. There are approximately 4 α2,3 sialyllactosemoieties coupled per antibody.

Example 22. Preparation of α2,6 Sialyllactose Maleimide by SilylatingLactose Maleimide and Conjugation to Hinge Disulfides of a RabbitPolyclonal Antibody Through α2,3- or α2,6-Linkages Resulting in HighSialylation

The conjugation of multiple copies of α2,6-sialylated glycans to thehinge disulfides of a rabbit polyclonal antibody was also investigated.Since the α2,3 sialyllactose maleimide was successfully produced using achemoenzyme approach (see above, Example 21), a similar method was usedto produce α2,6 sialyllactose maleimide (minor modifications of theprotocol included the use of a different sialyltransferase). To produceα2,6 sialylated glycan for conjugation, lactose maleimide (˜5 mg) wassialylated in vitro with 0.5 U of a bacterial α2,6 sialyltransferasefrom Photobacterium damsela in Tris buffer (pH 8) for 1 hr at 37° C.After enzymatic reaction, the product was applied to a QAE-sepharosecolumn. The column was washed with 10 fractions of 1 ml 2 mM Tris (pH8), 5 fractions of 1 ml of Tris buffer containing 20 mM NaCl, and 5fractions of 1 ml Tris buffer containing 70 mM NaCl. The aliquots fromeach fraction were analyzed using Dionex HPLC alongside lactose and α2,6sialyllactose standards. The oligosaccharide profiles of the standardsand one of the eluted fractions are shown in FIGS. 60 (A-D). Thefractions containing α2,6 sialyllactose maleimide were also analyzed andconfirmed by MALDI-TOF. The glycan in one of the fractions can be seenin FIG. 61.

The amount of α2,6 sialyllactose maleimide purified was then estimatedusing sialic acid quantitation analysis which indicated a recovery of˜1.5 mg sialyllactose maleimide. Once the glycan was prepared, theconjugation of antibody with either α2,6 sialyllactose maleimide or α2,3sialyllactose maleimide was tested using thiol chemistry. A rabbitpolyclonal IgG antibody (1 mg) was buffer-exchanged and reduced withTCEP at a 4 molar excess (over antibody) for 2 hrs at 37° C. The reducedantibody was then split in half: one portion was conjugated to 24 molarexcess of α2,6 sialyllactose maleimide, and the other to α2,3sialyllactose maleimide for 1 hr at room temperature. The two conjugateswere then buffer-exchanged into PBS before SDS-PAGE analysis (FIG. 62A)and sialic acid quantitation using Dionex HPLC (FIG. 62B). Sialic acidquantitation was was used to estimate the number of glycan conjugated.Aliquots of control antibody and thiol-conjugated antibody were treatedwith or without sialidase (1 U per mg) overnight at 37° C. beforesupernatants were recovered through filtration (10 kDa MWCO). The sialicacid content of the supernatants was measured and compared to samplestreated without sialidase (control). The analysis demonstrated thatapproximately 7 glycans (either α2,3- or α2,6-sialyllactose glycans)were conjugated to the polyclonal antibody by this method.

Example 23. PEGylation of NNAS Using GAM Chemistry

A mouse NNAS (S298N/T299A/Y300S) mutant monoclonal antibody wasgalactosylated and desialylated, generating a Gal NNAS monoclonalantibody without any protease degradation. This antibody was modifiedwith galactose oxidase (GAO) to generate galactose aldehyde. Thegalactose aldehyde was then conjugated with 2 or 5 kDa of aminooxypolyethylene glycol (PEG). FIG. 63 depicts the characterization ofcontrol and enzyme modified (desalylated/galactosylated) NNAS mutantantibodies using SDS-PAGE and lectin blotting. FIG. 64 depicts thecharacterization through reducing SDS-PAGE of the PEGylation of acontrol antibody and Gal NNAS with various amounts of galactose oxidase.These results demonstrate that Gal NNAS can be PEGylated efficientlywith significant amounts of mono-, bi-, and tri-PEG conjugated per heavychain. FIG. 66 depicts the characterization through reducing SDS-PAGE ofthe PEGylation of a control antibody and Gal NNAS with various molarexcess of PEG over antibody. Protein Simple scans characterizing thePEGylation of the antibodies demonstrate that approximately 1.5-1.7 PEGmoieties are conjugated per heavy chain (or about 3-3.4 PEG perantibody) (FIGS. 65 and 67).

Example 24. PEGylation of NNAS Using GAM Chemistry

An NNAS antibody was galactosylated with 50 mU/mg galactosyltransferaseand subsequently desialylated with 1 U/mg sialidase in 50 mM MES buffer(pH 6.5). Desialylated fetuin and NNAS as well as galactosylated NNASwere then treated with galactose oxidase (57 mU/mg)/catalase in thepresence or absence of 0.5 mM copper acetate before conjugation with 25molar excess of 5 kDa aminoxy PEG (FIG. 69A). In another experiment,galactosylated NNAS was treated with galactose oxidase (57mU/mg)/catalase in the presence of 0, 0.02, 0.1 and 0.5 mM copperacetate before conjugation with 25 molar excess of 5 kDa aminoxy PEG(FIG. 69B). Antibody oxidized with galactose oxidase in the presence ofcopper acetate showed a higher degree of PEGylation than the sameantibody reacted with galactose oxidase in the absence of copperacetate. Significantly higher levels of PEGylation were observed whenthe conjugation was performed in a reaction containing copper sulfate inconcentrations above 0.1 mM.

Example 25. Modification of Wild-Type and Mutant Herceptin UsingSialidase/Galactosyltransferase

Wild-type and mutant (A114N, NNAS, and A114N/NNAS) Herceptin antibodieswere enzymatically modified with 50 mU/mg galactosyltransferase andsubsequently desialylated with 1 U/mg sialidase in 50 mM MES buffer (pH6.5). The modified antibodies were analyzed using SDS-PAGE (reducing andnonreducing), lectin blotting with ECL (a plant lectin specific forterminal galactose), and terminal galactose quantitation using DionexHPLC analysis of released galactose by galactosidase (FIG. 70). Enzymemodified antibodies containing approximately three to nine terminalgalactose were obtained with the NNAS and NNAS/A114N double mutantsdemonstrating a higher level of terminal galactose than the wild-typeand A114N mutant.

Example 26. PEGylation of Wild-Type and Mutant Antibodies Using the SAMConjugation Method

Wild-type and (A114N, NNAS, and A114N/NNAS) Herceptin antibodies werePEGylated using sialic acid-mediated (SAM) conjugation. The antibodieswere subsequently oxidized with 2 mM periodate. After buffer exchange,the oxidized antibodies were PEGylated with 25 molar excess of 5 kDaaminoxy PEG. The sialic acid content of the wild-type and mutantantibodies was measured using Dionex HPLC (FIG. 71). The PEGylatedantibodies were then analyzed using reducing and non-reducing SDS-PAGE(FIG. 72). Further, the PEGylation (PAR, number of PEG per antibody) wasestimated by analyzing the scanned gels using ProteinSimple (FIG. 73).The NNAS, A114N, and A114N/NNAS mutants all showed higher PAR (2.7-4.6)than wild-type Herceptin antibodies (1.4).

Example 27. Investigation of Uptake of Glycoengineered Antibodies withGalactose Containing Glycan Ligands

A polyclonal antibody was either enzymatic modified withgalactosyltransferase (Gal Transferase), conjugated to lactose aminoxy(Gal-Glc to 297: conjugated to sialic acid in glycans from Asn-297 ofsialylated antibody), or conjugated to lactose maleimide (Gal-Glc toHinge: conjugated to cysteines in hinge disulfides). The control,modified, or conjugated antibodies were then incubated with HepG2 cells(a hepatocyte cell line expressing ASGPR) for 1-2 hrs at 37° C. beforethe uptaken antibodies were measured using Immunofluorescence staining(FIG. 74). The results showed increased HepG2 cell uptake of enzymaticmodified or lactose conjugated antibodies.

Example 28. Conjugation of a Trivalent GalNAc Glycan to Herceptin

Herceptin (anti-Her2) was sialylated and conjugated with a trivalentGalNAc glycan (FIG. 75) for targeting ASGPR using the SAM conjugationmethod. Subsequently, surface plasmon resonance experiments (Biacore)were used to asses the binding of these trivalent GalNAcglycan-conjugated antibodies to ASGPR subunit H1 (FIG. 76).

Example 29. Conjugation of Trivalent GalNAc Glycan and TrivalentGalactose Containing Glycopeptide to a Recombinant Lysosomal Enzyme

A recombinant lysosomal enzyme was conjugated with either trivalentGalNAc glycan or trivalent galactose containing glycopeptides (FIG. 77)for targeting ASGPR using the SAM conjugation method. Subsequently,surface plasmon resonance experiments (Biacore) were used to asses thebinding of these trivalent GalNAc glycan-conjugated and trivalentgalactose containing glycopeptide-conjugated enzymes to ASGPR subunit H1(FIG. 78). The results showed strong ASGPR binding of trivalent GalNAcglycan conjugated recombinant lysosomal enzyme. FIGS. 79(A-D) show anumber of exemplary trivalent GalNAc moieties. Examples are shown thathave no spacer, a 12 Å spacer, a 1 kDa (approximately 80 Å) spacer, andboth a ˜20 Å spacer and a 1 kDa (approximately 80 Å) spacer. A secondrecombinant lysosomal enzyme (rhGAA) was also conjugated with trivalentGalNAc glycan C12. FIG. 80 shows a graph depicting the results ofconjugation of periodate oxidized recombinant lysosomal enzyme rhGAAwith an excess of trivalent GalNAc glycan C12. The resulting glycan perGAA conjugated is depicted with 20, 40, 80, and 200-fold molar excessglycan over rhGAA. FIG. 81 depicts ASGPR binding of a recombinantlysosomal enzymes conjugated with trivalent GalNAc glycan C12 onBiacore. Enzymes conjugated with 20 (conjugate 1), 40, 80, and 200-fold(conjugate 4) excess of glycan all show strong binding to ASGPRsubunit 1. There is no significant difference in binding among theconjugates (conjugates 1 to 4).

1. A method of making a binding polypeptide comprising at least one modified glycan, wherein the glycan comprises at least one moiety of Formula (IV):

the method comprising reacting an effector moiety of Formula (I): NH₂-Q-CON—X  Formula (I), with a precursor binding polypeptide comprising an oxidized glycan wherein: A) Q is NH or O; B) CON is a connector moiety; C) X is a targeting moiety that binds to a receptor on a cell, such that the binding polypeptide is internalized by the cell; D) Gal is a galactose moiety; and E) Sia is a sialic acid moiety.
 2. The method of claim 1, wherein the targeting moiety binds to a mannose 6 phosphate receptor (M6PR) on a cell.
 3. The method of claim 2, wherein the targeting moiety comprises a mannose 6 phosphate (M6P) moiety, optionally wherein the M6P moiety comprises bis Man-6-P hexamannose.
 4. (canceled)
 5. The method of claim 1, wherein the targeting moiety binds to a sialic acid-binding immunoglobulin-type lectin (Siglec) on the cell.
 6. The method of claim 5, wherein the Siglec is selected from the group consisting of sialoadhesin (Siglec-1), CD22 (Siglec-2), CD33 (Siglec-3), myelin-associated glycoprotein (MAG (Siglec-4)), Siglec-5, Siglec-6, Siglec-7, Siglec-8, Siglec-9, Siglec-10, Siglec-11, Siglec-12, Siglec-14, and Siglec-15, optionally wherein the targeting moiety comprises an α2,3-, α2,6-, or α2,8-linked sialic acid residue.
 7. (canceled)
 8. (canceled)
 9. The method of claim 1, wherein the targeting moiety binds to a C-type lectin receptor, a galectin, or an L-type lectin receptor, optionally wherein the targeting moiety binds to an asialoglycoprotein receptor (ASGPR) on a cell.
 10. (canceled)
 11. The method of claim 9, wherein the targeting moiety comprises a GalNAc moiety, optionally wherein the targeting moiety is a trivalent GalNAc glycan moiety, and optionally wherein the targeting moiety is a tri-galactosylated glycopeptide or lactose₃-Cys₃Gly₄.
 12. (canceled)
 13. (canceled)
 14. The method of claim 1, wherein the binding polypeptide comprises 1, 2, 3, 4, or more binding sites responsible for selectively binding to a target antigen of interest.
 15. The method of claim 14, wherein at least one binding site comprises a ligand binding site of a receptor, or a receptor binding site of a ligand, optionally wherein the ligand is selected from the group consisting of a polysaccharide, a sugar molecule, a carbohydrate.
 16. (canceled)
 17. The method of claim 1, wherein the binding polypeptide is an antibody or antigen fragment thereof, the antibody comprises a single chain variable region (ScFv) sequence, the binding polypeptide is an antigen binding fragment, or the binding polypeptide comprises one binding site comprising an antibody variable domain.
 18. (canceled)
 19. The method of claim 17, wherein the antibody or antigen binding fragment thereof is bispecific, optionally wherein the bispecific antibody is a cross-over dual variable domain IgG (CODV-IgG) bispecific antibody; or trispecific. 20-23. (canceled)
 24. The method of claim 1, wherein the cell is a mammalian cell, optionally selected from an immune cell, a liver cell, a tumor cell, a vascular cell, an epithelial cell, or a mesenchymal cell; or a B cell, a T cell, a dendritic cell, a natural killer (NK) cell, a macrophage, a neutrophil, a hepatocyte, a liver sinusoidal endothelial cell, or a hepatoma cell.
 25. (canceled)
 26. (canceled)
 27. The method of claim 1, wherein: (a) the amount of the binding polypeptide internalized by the cell is greater than the amount of a reference binding polypeptide lacking a targeting moiety internalized by the cell, (b) Q is O, or (c) the glycan comprises at least one moiety of the following structural formula:


28. (canceled)
 29. (canceled)
 30. The method of claim 1, wherein the binding polypeptide comprises an Fc domain, and wherein: (a) the modified glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 297 of the Fc domain, according to EU numbering, (b) the modified glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 298 of the Fc domain, according to EU numbering, or (c) the Fc domain is human. 31-33. (canceled)
 34. The method of claim 1, wherein the binding polypeptide comprises a CH1 domain, and optionally wherein the modified glycan is N-linked to the binding polypeptide via an asparagine residue at amino acid position 114 of the CH1 domain, according to Kabat numbering.
 35. (canceled)
 36. The method of claim 1, wherein: (a) the connector moiety comprises a pH-sensitive linker, a disulfide linker, an enzyme-sensitive linker or another cleavable linker moiety, (b) the ratio of targeting moiety to binding polypeptide is equal to or more than about 4, (c) the ratio of targeting moiety to binding polypeptide is at least about 2, (d) the precursor binding polypeptide is generated by oxidizing an initial binding polypeptide comprising at least one glycan moiety of the following structural formula:

(e) the precursor binding polypeptide is generated by oxidizing an initial binding polypeptide comprising at least one glycan moiety of the following structural formula:

(f) the oxidized glycan of the precursor binding polypeptide comprises at least one moiety of the following structural formula:

or (g) the oxidized glycan of the precursor binding polypeptide comprises at least one moiety of the following structural formula:

37-42. (canceled)
 43. The method of claim 1, wherein oxidized glycan of the precursor binding polypeptide is generated by reacting an initial binding polypeptide comprising a glycan with a mildly oxidizing agent.
 44. The method of claim 43, wherein: the mildly oxidizing agent is sodium periodate, optionally wherein less than 2 mM sodium periodate is employed, or the mildly oxidizing agent is periodate or galactose oxidase.
 45. (canceled)
 46. (canceled)
 47. The method of claim 44, wherein the reacting step is conducted in the presence of a salt comprising a metal ion, optionally wherein the metal ion is a copper ion, the salt is copper acetate, or the salt comprising a metal ion is present at a concentration of at least 0.1 mM. 48-50. (canceled)
 51. The method of claim 1, wherein the precursor binding polypeptide comprises one or two terminal sialic acid residues, optionally wherein the terminal sialic acid residues are introduced by treatment of the binding polypeptide with a sialyltransferase or combination of sialyltransferase and galactosyltransferase.
 52. (canceled) 